Methods in Cell Biology 2015
Johannes A. Schmid
Internet: www.meduniwien.ac.at/user/johannes.schmid
some contents contributed by:
Dr. Lukas Mach
Institut für Angewandte Genetik und Zellbiologie ,
Univ. f. Bodenkultur
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Overview of Topics
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scientific strategies and assay systems
cell culture
labelling and transfection of cells
analysis of cellular components
analysis of molecular interactions
fluorescence measurements
microscopy
flow analysis (fluorescence activated cell sorting, FACS)
analysis of various cellular processes (proliferation, apoptosis..)
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Details of the lecture
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Scientific strategies
cell culture
labelling and transfection of cells
a) radioactive and chemical labelling
b) transfections: overexpression of genes and gene suppression
c) reporter gene assays
gene-suppression (siRNA-technologies)
analysis of DNA and proteins (electrophoresis and blotting)
subcellular fractionation (centrifugations…)
methods to detect macromolecular interactions
a) Yeast 1- und 2-hybrid systems
b) co-immunoprecipitations
c) fluorescence resonance energy transfer (FRET)
methods of fluorescence measurements
realtime-PCR
transmitted light microscopy and contrast principles
fluorescence microscopy
confocal laser scanning microscopy
flow analysis (FACS)
analysis of various cellular processes (proliferation, apoptosis..)
methods to investigate vesicular transport processes
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Nodes of regulation in cellular systems
cell membrane
ligands
receptors
oligomerization
transport
signal transduction
signal
transduction
modified
protein
Golgi
transport
transcription
factor
activation
posttranslational
modification
ER
protein
transport
nucleus
poly-ubiquitination
degradation
translation
transcription
splicing
pre-mRNA
mRNA
mRNA
transport
DNA
micro-RNA
processing
pre-micro-RNA
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micro-RNA
degradation
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General scientific strategies
• descriptive strategies: the whole system (cell, organism) is
observed without influencing it
advantage: physiological states are not altered
disadvantage: it is difficult to elucidate cause-effect relationships
• mechanistic (manipulating) strategies: various factors are kept
constant, while others are altered on purpose – the change in
the whole system is monitored.
advantage : cause-effect relationships can be monitored or
detected
disadvantage : the physiological steady state is altered and
influenced. Results might be artefacts of the measurement
system.
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Experimental Systems I
• In vitro, biochemical systems: investigation in a solution
(e.g. enzyme reactions): many parameters can be fixed
(temperature, pH, buffer composition…).
Example: enzyme activity assay, EMSA …
• In vitro, cell biological systems: including cellular structures
e.g. in vitro-transcription/translation with membrane
components, in vitro-fusion of endosomes, nuclear import
assays with isolated nuclei etc. Experimental conditions
have to be set in a way that cellular structures are not
damaged (e.g. isotonic buffer, physiological pH….); reaction
partners can be influenced widely (e.g. antibodies can be
added…)
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Experimental Systems II
• cell culture systems
- immortalized cell lines: unlimited passages
- primary cells: limited passage number, more
complicated cell culture (e.g. coated plates…)
- co-culture of different cell types:
e.g. keratinocytes + fibroblasts in a collagen matrix.
• xenograft systems:
- cells are injected into immuno-compromized mice
(nude mice, SCID mice); e.g. subcutaneously
- tissue recombination systems (e.g. prostate epithelial
cells with mesenchymal cells injected into the renal
capsule of SCID mice)
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Experimental Systems III
• Organ cultures
(e.g. skin sheets,
brain slices…)
• Organ perfusions
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Experimental Systems IV
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Animal Experiments:
Just the whole organism provides the full complex biological system that is
relevant for most biomedical research topics. The whole organism includes
superordinated systems such as the nervous system, the blood circulation,
the endocrine system and so on. The cells are in their normal organ
environment; cellular communications are intact…. – thus the highest
possible physiological state can be achieved. However, specific components
of the system (e.g. certain cells) are not easily accessible – and specific
experimental manipulations (e.g. of specific cells without side effects) are
often difficult). Cause-effect relationships are often difficult to elucidate –
and there is a big „black box“ due to the complexity of the system.
It has to be considered that results obtained with animals such as mice often
cannot be transferred to human beings despite the highly physiological
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system.
Experimental Systems V
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Special case: Transgene and knock-out animals :
Specific elimination of genes (knock-out) or incorporation of genes
(transgenes, knock-in) allow a better elucidation of cause-effect relationships.
However, classical knock-outs (eliminating a gene in all cells) – is often
embryonically lethal – and does not allow conclusions for its function in the
adult animal (e.g. mouse). Vice versa, it can happen that there is no
phenotype (if the function of the gene is taken over by another gene). Knockins can be quite artificial as well (if the transgene is expressed at higher levels
with strong promoters). Modern approaches often aim for „conditional knockouts“ or knock-ins: In most cases the Cre-recombinase / loxP system is used
for that purpose:
Conditional knock-out: the gene (or a crucial exon) is placed between loxP
sites – Cre recombinase (which can be expressed in specific organs or cells by
organ specific promoters) cuts out the gene – thus the gene is deleted just in
a certain organ or cell type; using inducible Cre, this system allows gene
deletion at a defined time point (e.g. when the animal is adult).
Conditional knock-in: the gene is placed behind a Stop-cassette, which is
flanked by loxP sites. Without Cre activity, the gene is not expressed, with Cre
activity (organ specific), the Stop cassette is excised and the gene is expressed
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Conditional transgene mouse models with the
Cre / loxP system
1. Cre mouse strain
2. loxP-mouse strain
• Cre recombinase cuts out
sequences between loxP sites
(or inverts sequences between
inverted loxP sites)
• Cre expression can be
rendered cell-type or organspecific using cell-type specific
promoters driving Cre
expression (spatial control)
• Cre expression can be made
inducible by using chimeras of
Cre with mutated estrogen
receptor domains
(temporal control:
e.g. Cre-ERT2)
• Endogenous genes can be
flanked by loxP sites (using
recombination techniques –
e.g. affecting essential exons)
> conditional knock-out
• Genes can be overexpressed
conditionally by inserting an
expression construct headed
by a loxP-flanked „Stop
cassette“, which is cut out by
Cre recombinase
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Examples for conditional mouse models
cell-type specific
promoter
mER
Cre
mER
Cre-ERT2
mER: mutated estrogen receptor (responds to tamoxifen as ligand; without
the ligand it keeps the Cre in the cytosol > inactive; upon addition of
tamoxifen, the nuclear localization sequences of mER become active leading
to translocation of the chimera into the nucleus, where Cre recombinase can
exert its function on loxP-flanked DNA sequences).
conditional knock-out
conditional transgene
Gene of interest
loxP
Stop
loxP
Gene of interest
good promoter
(e.g. pCAGGS)
mER
Tam
Cre
mER
mER
Tam
Tam
Cre
mER
Tam
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Specific cell ablation or cell labeling
in transgenic mice
loxP
loxP
Stop
DTR (Diphteria toxin receptor)
good promoter
(e.g. pCAGGS)
cross-breeding with a cell-type specific Cre
strain
DTR is expressed only in specific cell types
injection of diphteria toxin leads to specific
killing of these cells
loxP
loxP
Stop
good promoter
(e.g. pCAGGS)
EYFP (enhanced yellow fluorescent protein)
> fluorescent labeling of a specific cell type of interest
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Genome Editing for generating transgene
animals
• Novel methods to edit genes directly in the genome, e.g. using
CRISPR/Cas9 technology or Zn-finger nucleases, or TALENs
allow faster and even multiplexing type manipulations (e.g.
targeting 5 genes simultaneously)
• Genome editing is then usually done in ES-cells, which are
subsequently injected into blastocysts.
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Cell Culture Methods
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cell culture of immortalized cells
cell culture of primary cells (often just a few passages)
culture of polarized cells
co-culture of different cell types
primary fibroblasts
(from skin dermis)
transformed
fibroblasts
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What you need for cell culture
•
Incubator (37°C) with CO2supply and humidification
•
Sterile bench (Laminar Flow: a
laminar flow of filtered air keeps
the bench sterile) – has to be
switched on approx. 10 min
before using it, the filter has to
be checked from time to time
(Particle Measurement)
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inverted cell culture microscope
(4x, 10x, 20x objectives)
•
L2-biosafety (e.g. for viruswork): not only incoming air is
filtered, but also the air that
leaves the laminar flow.
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Culture Media
mammalian cells need media with vitamins, amino
acids, hormones and growth factors
Serum, mostly fetal calf serum (FCS), is the source of
growth factors (such as FGF, EGF…)
Common composition
- DMEM (Dulbecco‘s Modified Essential Medium)
- 10% FCS
- 2 mM Glutamine (unstable amino acid, has to be added
again, if the medium is older than app. 6 weeks)
- Penicillin (100 u/ml)
- Streptomycin (100 µg/ml)
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Fetal Calf Serum (FCS)
has high concentrations of growth factors
(e.g.: EGF, epidermal growth factor; FGF,
fibroblast growth factor; IGF, insulin-like
growth factor)
low amount of antibodies → compatible
with cells of other species
complement system is inactivated by a
heat shock (30 min., 56 °C)
disadvantage of FCS: expensive (≈ 100 €
per Liter)
might contain contaminants such as
tetracyclin (important if you use a Tetinducible cell culture system > purchase
guaranteed Tet-free FCS)
Alternative sources of serum: normal calf
serum, horse serum…
Special Growth
Factors
Nerve- Growth Factor
(NGF):
for neuronal cells
Hepatocyte-Growth
Factor (HGF): induces cell
division of hepatocytes
Keratinocyte-Growth
Factor (KGF): for culture
of skin epithelial cells
(keratinocytes)
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Adhesion factors
adherent cells versus suspension cells
cell culture dishes: usually made hydrophilic
(charged groups) – sufficient for most cell types
collagen; Gelatine (denatured collagen)
sometimes necessary for good attachment (e.g. for
primary endothelial cells)
Components of extracellular matrix:
Fibronectin, Laminin – often better than collagen.
special case: „Feeder“-cells (irradiated) – or coculture of cells (e.g. fibroblasts in a collagen matrix
with keratinocytes on top)
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Contaminations in cell culture
Bacteria: might be a problem, when
they are resistant against the antibiotics
that are used (mostly Pen/Strep) >
then you have to use other antibiotics
(e.g. kanamycin)
Mycoplasm: procaryotes of an ancient
evolutionary stage, which do not have a
normal bacterial cell wall (therefore
they are resistant against Penicillin)
(can be eliminated with Kanamycin)
Yeast: rare (might occur when yeast
and mammalian cell culture are not
strictly separated)
Fungi: quite rare
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Unnoticed mycoplasm contaminations can screw up experimental results
> Tests for Mycoplasm-Contamination
DNA-staining with DAPI or Hoechst 33258
no Mycoplasm
Mycoplasm
Alternative:
PCR- detection of Mycoplasm-DNA
(commercial kits are available)
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Cell culture of polarized cells
apical side
tight junction
lateral side
the cells have to build the
tight junctions for building up
the polarity.
Often they are cultivated on
filters, where the two sides
are accessible.
basal membrane
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Methods to investigate polarized cells
Model systems
1. simple cell culture: just one side of the cell is accessible.
2. cell culture on microporous membranes: both sides are
experimentally accessible, the establishment of a tight
polarized monolayer can be checked be measuring the
electrical resistance between the two sides.
3. Organ cultures: e.g. Living Skin Equivalent
4. Organ perfusion: e.g. perfusion of isolated rat liver
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Culture of polarized cells on membranes
Measurement of electrical
resistance
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Cultivation of polarized cells on electrode
chambers
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Cultivation of polarized cells on electrode
chambers
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Organotypic culture: Example: Skin Equivalent
Keratinocytes (cells of the upper layer of skin, the epidermis) are
seeded onto a collagen matrix, which contains fibroblasts (dermis
cells). As soon as they build a monolayer, they are elevated to the
surface of the medium (with their upper side exposed to air) – this
induces cell differentiation and the formation of a pseudo-epidermis
with several cell layers.
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Perfusionsdruck
(cm H 2(cm
O)
perfusion pressure
Pump
Peristaltikpumpe
H2O)
Liver
LEBER
bile canula
Gallengangskanüle
Thermosensor
V.
porta
V. porta
Kanüle
cavaKanüle
canula
V.V.cava
Computer
Fraktionskollektor
fraction collector
gas humidification
Gas-Befeuchtung
temperature
Temperatur-
recording
Messung
Schreiber
Organ Perfusion
Polarized cells such as hepatocytes
can be maintained in their organ
architecture maintaining their
polarity. The organ is perfused
using glas capillaries linked to the
normal blood vessels the supply
the organ with blood (and
nutrients/oxygen). A buffer (37°C,
percolated with O2) containing the
nutrients can be perfused through
the liver. Marker substances can
be applied and their transport
from the blood side (basolateral)
to the bile side (apical) can be
determined.
Wasserbad
Water Bath
Perfusionspuffer
Perfusion
Buffer
93% O 2
7% CO 2
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Example of an experiment with polarized cells
Transmigration of Leukocytes through a Monolayer of polarized
endothelial cells
Leukocytes are labeled with a
fluorescent marker (e.g. CFSE
– an ester, which is turned
fluorescent just after uptake
into cells due to esterases)
Cells are seed onto a layer of endothelial cells (e.g. after activating the
endothelial cells with inflammatory cytokines – which leads to the
synthesis of adhesion molecules on the surface of the endothelial
cells). Adhesion of leukocytes leads to transmigration into the lower
chamber. The extent of the transmigration can be determined by lysing
the cells and quantifying the fluorescence (or by counting the
fluorescent cells)
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Methods for labeling of cellular components
• Radioactive labeling: e.g. with radioactive amino acids (35SMethionin…), which are incorporated into newly synthesized
proteins. Pulse/chase experiments can give insights into half life,
transport. Processing… of proteins.
• Internalisation of high molecular weight markers (loading of
endosomes and lysosomes).
• Labeling of cell surface proteins (e.g. by biotinylation with cellimpermeable reactive biotin compounds).
• Addition of cell-permeable labeled substances, which integrate into
specific structures (e.g. Golgi-specific fluorescent lipids)
• Transfection of cells with expression plasmids
• Protein-Transduction (cell-permeable peptides)
• Micro-Injection of substances.
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Radioactive Labeling of Biomolecules
Amino acids:
[35S]Methionine, [3H]Leucin
Monosaccharides: [3H]Mannose, [3H]Glucosamine
Phosphate:
[32P] phosphoric acid
Alternative:
33P:
lower radioactivity
Sulfate:
[35S]Sulfuric acid
Fatty acids:
[3H]Palmitinic acid
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Radionuclides
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Metabolic labeling with amino acids
usually [35S]Methionine labeling
advantage: high specific radioactivity
disadvantage: relatively rare amino acid > check first,
how many methionine are in the protein! (e.g.
Ubiquitin: only 1 Methionine, but 9 Leucines)
requires Methionine-free culture medium for depletion
of endogenous stores by pre-incubation
(addition of dialyzed FCS)
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Metabolic Labeling with Monosaccharides
[3H]Mannose or [3H]Glucosamine
Activated in the cytosol by coupling to nucleotides →
GDP-Mannose, UDP-N-Acetyl-Glucosamine (UDPGlcNAc)
requires Glucose-poor culture medium
Addition of alternative energy sources
(Glutamine, Pyruvate)
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Pulse-Chase-Experiments
preincubation without marker (depletion of
endogenous stores)
Addition of the labeled substance → "Pulse" (5 min 1 h or longer)
Stop of the "Pulse" by addition of an excess of
unlabeled compound
further incubation → "Chase" (min - 48 h)
Stop of culture → Analyses
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Example for a Pulse-Chase-Experiment
Fibroblasts of patients, were labeled with
35S-methionine:
Pulse: 1 h at 37°C
Chase: 7 h at 19°C and 37°C (19°C inhibits transport from trans-Golgi to
late endosomes/pre-lysosomes) > Immunoprec. and autoradiography
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Iodination of Proteins (Labeling with 125J)
direct: modification of Tyrosine with [125J]:
- chemical oxidation: Chloramine-T oxidizes Na125J
and leads to iodination of Tyrosine
- enzymatic: Lactoperoxidase: oxidizes Na125J in
presence of H2O2.
indirect: Modification of amino groups (Lysine, NTerminus) with [125J]-labeled Bolton-Hunter-Reagenz
(N-succinimidyl 3-(4-hydroxy 5-[125J]iodophenyl)propionate)
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Biotinylation
High affinity binding partner of Avidin and
Streptavidin (> easy purification and
detection by beads, coated ELISA-Plates…)
Streptavidin has 4 binding sites for biotin
(complexes can be formed with bivalent
biotin-linkers!) > signal amplification is
possible (ABC: avidin-biotin-complexes)
Most commonly used method to label cell
surface proteins
Biotinylation of amino groups with BiotinHydroxysuccinimid-Esters or of cysteines
(SH-group) by Maleimide-derivates
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Labeling of Endosomes, Lysosomes
• By high molecular weight compounds, which are easily
detectable, not permeable for the cytoplasmic membrane and
taken up by endocytosis
Examples:
a) unspecific internalization:
- FITC-Dextran (Fluorescein-labeled)
- Enzymes (Peroxidase…)
b) Specifically by receptors
Pulse/Chase conditions can be used to labeled specifically early
or late endosomes or lysosomes (e.g. using temperature
blocks....)
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Transfections
Usually designates the incorporation of DNA into mammalian cells. DNA present
in form of plasmids.
Transient Transfection: plasmid remains outside of the genome and is slowly
lost (degradation, dilution by cell division), exception: episomal replication –
e.g. SV40-Plasmids in COS-cells). The transfection efficiency varies – but can
reach close to 100%
Stable Transfection: integration of foreign DNA into the genome (Efficiency:
usually below 0.1%). Isolation of stably transfected clones requires selection
genes (for antibiotic resistance, e.g. puromycin, G418…). Plasmids are usually
linearized before transfection to increase the possibility of correct integration.
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Example for a mammalian expression plasmid
(Replication origins no shown)
CMV-Promoter
poly-adenylation signal
selction gene
(bacterial
selection with
Kanamycin,
mammalianselection with
G418)
reporter gene
Multiple
Cloning Site
target gene
SV40-Promoter
suited for linearization
poly-adenylation signal
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Selection markers for stable transfections
•Aminoglycoside-Phosphotransferase: Resistance against Neomycin
(bacteria) und G418 (mammalian cells). Selektion with G418 takes
quite long (app 2 weeks). Surviving colonies are isolated and further
cultures under selection pressure.
•Hygromycin-Phosphotransferase
•Puromycin-Acetyltransferase
•Dihydrofolat-Reductase (DHFR): Selection with Methotrexat; allows
amplifications of the target gene.
•GFP-Fusion proteins: fluorescence
can be used for selection: example:
EGFP-NF-κB expressing CHO-cells
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Chemical Transfection Methods
Ca2+
• DNA-Calcium precipitates: at exact pH and
Ca2+-concentration: High efficiencies with
293-cells (90% and more), expression levels
are usually moderate.
Ca2+
Ca2+
• Liposome mediated transfection: kationic
lipids which bind the negatively charged DNA,
and which are taken up as liposomes
Quite high transfection efficiencies but also
sometimes toxic effects and potentially
artefacts by high expression levels or
alteration of cellular membranes. (e.g.
Lipofectamine, Fugene...)
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Buffers
• HeBS-Buffer (Hepes buffered saline)
8 g NaCl - 280 mM final concentration
0.2 g Na2HPO4.7H2O (or 0.107 g anhydrous) - 1.5 mM
6.5 g Hepes (Sigma H-7006) (or 5.96 g of free acid) - 50 mM
400 ml A.dest.
Adjust the pH to exactly 7.05 (calibrate pH-meter with pH 4.01 and pH 7.00 buffers before). Add A.dest. to 500 ml,
filter through 0.2 µm filters and store in aliquots at -20°C (not longer than 6 months). Thawed aliquots shouldn't be
frozen again.
• CaCl2: 29.4 g CaCl2.2H2O (MW=147) in 100 ml A.dest (final conc.: 2 M) Filter through 0.2 µm filters and store
aliquoted at -20°C.
• Chloroquine (optional): chloroquine. 2H2O (Sigma C-6628): 12.9 mg/ml in PBS (conc.: 25 mM). Filter through
0.2 µm filters and store at -20°C.
Protocoll: Calcium-Transfection
Procedure (amounts are given for 6-wells):
1. Seed cells (about 500 000 cells per 6-well = per 10 cm2 ) one day before the transfection (in DMEM/10% FCS)
2. (Optional: 1 h before transfection, exchange the medium for medium containing 25µM chloroquine)
3. Thaw HeBS and CaCl2 at room temperature
4. For each transfection prepare aliquots of 71 µl HeBS
5. Prepare the DNA/ CaCl2-Mix: 4 µg DNA (total) in 62 µl A.dest. + 9 µl CaCl2
6. Add the DNA/ CaCl2-Mix drop-wise to the HeBS aliquots (by screwing the Gilson pipette) and slightly mix after
each drop. Incubate for 2 - 3 min at R.T. to form the DNA-precipitate (not longer).
7. Add the DNA-precipitate drop-wise to the cells (by screwing the Gilson pipette and moving it to cover the whole
surface of the cell culture; don't swirl the dish).
8. Carefully transfer the dish back to the incubator. Incubate for 24 h (or in the presence of chloroquine: for 10 h)
and exchange the medium afterwards. (The transfection is in the presence of FCS!). The efficiency of transfection is
in the range of 70-90% for 293 cells. Harvest the cells after 48 h. The protocol is adapted from Neil Perkins who
adapted it from Gary Nolan in 1995
(See web site: http://www.stanford.edu/group/nolan/ or CP in Mol.Biol. 9.1 and 9.11.2-3)
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Lipofectamine2000 - Standard conditions
Transfection in presence of serum (e.g. 24h – 48h)
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Chemical Transfection Methodes II
• Dendrimeres: charged Polymeres that bind
DNA (e.g. Superfect / Qiagen)
• complexes with DEAE-Dextran (polycationic
Dextran)
• poly-ethylene-imine (PEI):
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Physical Transfection Methods
• Electroporation: common with
suspension cells,
electrocuvette
requires much DNA
Special case: AmaxaNucleofection (now Lonza)
• Particle-gun (“Gene Gun”):
DNA on gold particles shot by
pressure onto cells (e.g.
neurons in brain slices).
• Micro-Injection of DNA: into
single cells (just limited cell
number can be targeted).
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Viral Transfection Methods
• Adenoviruses, Retroviruses,
Lentiviruses:
Viruses developed fancy mechanisms to
get into cells – these are applied for
gene transfer. Virus-constructs are
generated, which contain the target gene
but not genes for virus replication (genes
essential for generating the viral particles
are supplied by “Packaging Cells”).
• Adenoviruses: transient Expression
• Retroviruses: stable integration but
target just proliferating cells
• Lentiviruses: stable integration, also
transduce quiescent, non-proliferating
cells.
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Adenovirus
Retrovirus
•Episomal gene expression
•Long-term, stable gene expression; inheritable
•Infects dividing &
nondividing cells
•Infects dividing cells only
•High-level protein
expression
•Moderate protein expression
•Viral titers of up to 1012
pfu/ml
•Viral titers of up to 106 cfu/ml (Can be
concentrated to 109 cfu/ml)
•Accommodates inserts of up
to 8 kb
•Accommodates inserts of up to 6.5 kb
•Elicits immune reactions in
vivo
•Does not elicit immune reactions in vivo
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Example of and
adenoviral system
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Adeno-associated virus (AAV)
•
•
•
•
•
is a small virus which infects human cells
is not causing any obvious disease
causes a very mild immune response
can infect both dividing and quiescent cells
AAV vectors persists mostly in an extrachromosomal state
without integrating into the genome of the host cell (the native
virus can integrate to some extent into the host genome).
• Promising gene therapy vectors (clinical trials have been done
for CFTR, hemophilia B, arthritis …)
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Protocol:
Production of retrovirus by transfection of packaging cells
Packaging cell line: Phoenix cells. These cells must not be too confluent since this will lower the
production of virus significantly.
Day 1
Plate phoenix cells on 15 cm plate (1.5-1.75 x 107 cells)
Day 2
((Transfect phoenix cells using 25 µg DNA and 75 µl Fugene6 + 1.8 ml OptiMEM.
Incubate 30 min RT and add to cells.)) OR better use CaPO4!!!
Day 3
Carefully remove old medium and add 25 ml of fresh medium.
Incubate at 32°C for 24h or 48h (or 72h).
Day 4-5
Harvest supernatant after 24-48h. Virus sup can be harvested until the cells start looking unhealthy.
Put supernatant in 50 ml tube in ice bucket in the hood. Add 22 ml fresh medium to the packaging
cells and put back into incubator.
Spin viral sup to pellet any remaining cells.
For storage of virus: transfer to cryotube and snap freeze with N2 store at -80°C. Upon freezing virus
titer goes down roughly twofold!
For use straight away: Filter virus sup through a 0.45-µm cellulose acetate or polysulfonic filter
(do NOT use nitrocellulose filter since it binds proteins in the retroviral membrane!). Keep on
ice until use.
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Protocol II:
Infection of MEFs with recombinant retrovirus for
making stable cell lines
Day 3
Split the target cells into 10 cm dishes.
Day 4
Add 8 µg/ml polybrene to the filtered viral supernatant. Mix gently by
inversion.
Replace the media in the target cells with the viral sup + polybrene.
Incubate at 37°C for 6h and add equal amount of media containing
polybrene.
Day 5
Replace the media.
Day 6
Replace the media with fresh media containing pyromycin to select for
transfectants. The amount of pyromycin used is determined by killing curve
experiments. Select colonies of stable transfectants keeping the pyromycin
in the media.
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Reporter-Gene Assays
Enzymes or other molecules, which are easily detectable, are
applied as „reporter molecules“ to detect promoter activities or the
activities of signaling pathways.
The reporter gene is cloned into an appropriate plasmid (e.g.
mammalian expression vector) and transfected into the cells of
interest, followed by the biological experiment (e.g. stimulation).
Examples for reporter-genes:
•
•
•
•
•
EGFP (Enhanced Green Fluorescent Protein) and variants thereof (e.g.
destabilized EGFP)
Luciferase
CAT (Chloramphenicol-Acetyl-Transferase)
ß-Galactosidase (lacZ)
SEAP (secreted alkaline phosphatase) etc.
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Regulated Promoters in Reporter Gene Assays
(artificial promoters)
•
Tandem Repeats of transcription factor binding sites (e.g. 5x NF-kappa
B). Often commercially available (e.g. from Stratagene or Clontech)
> can be used to determine the activation of a certain transcription
factor (or signaling pathway)
minimal
promoter
element
Reporter Gene
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Regulated, Natural Promoters in Reporter Gene
Assays
• Natural promoters usually contain binding sites for several
different transcription factors (sometimes several copies of a
single TF binding site) > are regulated by several signaling
pathways.
Example:
NFκB
IL-8
Promoter
IRF-1
GRE
AP-1
NF/IL-6
Luciferase
p65 / c-Rel
> can be used to determine the regulation of a specific promoter of interest
Luciferase is usually easier to measure with high sensitivity than the gene of
interest (in this case IL-8)
58
Normalization constructs with constitutive promoters
…usually used for the normalization control in reporter gene assays
(to compensate for differences in transfection efficiency, extraction efficiency
or viability of cells in different samples)
Usual setting: Pathway-specific reporter construct (e.g. with firefly luciferase)
+ constitutive normalization construct (e.g. Ubiquitin-Promoter driven βgalactosidase or Renilla-luciferase) > quantification of normalized values
(Luciferase / β-galactosidase or Firefly / Renilla-luciferase)
Promoters for the normalization vector:
•
•
•
•
•
CMV: from human Cytomegalo-Virus: induces strong constitutive
expression (fast, about 1 day after transfection)
RSV: from Rous Sarkoma-Virus: weaker, but very constant, constitutive
expression, slightly slower (takes 2 d)
SV40: Simian Virus 40-Promoter
Actin-Promoter: human promoter of a „housekeeping gene“
Ubiquitin-Promoter: human promoter of a „housekeeping gene“
59
29
Example of an Reporter Gene Assay
fold of control vector
Luciferase/β-Gal
50
46.36
45
EP-cells
40
DU145
35
26.66
30
25
20
15
10
5
1.94
4.63
0.90
0.49
0
p53
Rb
E2F
Activities of different signaling pathways or molecules (p53, Rb, E2F) are
assessed with reporter constructs containing respective transcription
factor binding sites upstream of a luciferase vector.
For normalization purposes a constitutively expressed control gene has
to be cotransfected (e.g. β-galactosidase downstream of a constitutive
promoter e.g. ubiquitin-promoter).
Values are calculated as Luciferase/β-Gal.
60
Reporter-Gene Assay Systems for Analyses of
Signaling Pathways
Transcription factor construct
Gene of interest
Transcription factor construct
ReporterPlasmid
In case that the gene of interests
activates the specific pathway,
which leads to phosphorylation
and activation of the used
transcriptionfactor construct, the
expression of the reporter (e.g.
luciferase) is induced.
ReporterPlasmid
61
30
Detection of reporter genes: GFP (and variants)
• Fluorescence measurement by
fluorometry (e.g. in 96-well
fluorescence readers)
• Microscopy
• Flow analysis (cytometry,
FACS: Fluorescence activated
cell sorting: GFP-containing
cells can be separated from
other cells and if necessary
also further cultivated after
purification)
GFP fluorescence >
62
Detection of Luciferase (Firefly or Renilla Luciferase)
•
Luciferin
LuciferaseZellextrakt
ATP
Very sensitive detection in cell extracts by measuring the
luminescence generated from luciferase in presence of
luciferin and ATP. The substrate Luciferin has to be injected
into the sample, and measured immediately (e.g. by
integrating for 5 sec) as the emitted luminescence decays
quickly. Emitted photons are measured with photomultiplier
tubes.
PMT
Measuring devices: Luminometer
(also as 96well devices available)
PMT: from http://www.molecularexpressions.com
63
31
Detection of β-Galactosidase (lacZ)
• often by photometry (e.g. in ELISA
readers) using a yellow substrates, which
turns red in presence of Galactosidase
(substrate: CPRG, Chlorophenolred-β-DGalactopyranoside). Detection at 595 nm.
• Alternative: Chemiluminescence
measurement
• Alternative: Fluorimetric Detection:
Substrate: e.g. 3-carboxyumbelliferyl
beta-D-galactopyranoside, CUG is
converted to a fluorescent product
• Microscopical detection: with X-Gal (5Bromo-4-chloro-3-indolyl-ß-D-galactoside)
as substrate: produces a dark blue
reaction product
64
β-Galactosidase Assay with CPRG
Lyse cells
(recommended cell lysis buffer: 0.25M Tris/HCl pH 8.0, 0.25% (v/v) NP40,
2.5 mM EDTA)
Pipet about 10 µl of extract into a 96-well plate (appropriate neg. control / blank: 10 µl of mock
transfected cells, or non-transfected cells – as there is a slight endogenous b-Gal activity) – leave one
well empty for blank (A-1)
Add 100 µl substrate solution to the wells (also to the blank-well)
Incubate until red color develops (min to hours – depending on b-Gal activity, if you have low activity
you can also incubate at 37°C)
Optional: Stop with 50 µl of Stop solution (only necessary if you want to time it exactly, e.g. by adding
the substrate in a timed way and stopping the reaction in the same way)
Measure with ELISA Reader at 570 nm (Filter #3) optimum: 595 nm
Lysis Buffer:
0.25M Tris/HCl pH 7.4 (or better 8.0)
0.25% (v/v) NP40
2.5 mM EDTA
CPRG-substrate solution: 1 mg/ml (= 1.65 mM)
in PBS + 10 mM KCl, + 1 mM MgCl2
alternative substrate buffer:
60 mM Na2HPO4 pH 8.0, 1 mM MgCl2, 10 mM KCl, 50 mM Mercapto-ethanol
65
Stop solution: 0.5M Na2CO3
32
Methods to suppress gene expression
(RNA interference)
Antisense-Technologies: to suppress gene expression were started many years
ago. Early approaches used antisense-oligonucleotides – but the effect was very
variable.
Alternative approaches used long antisense-strands hybridizing to the mRNA:
This usually leads to downregulation of gene expression – but quite often not
only for the targeted gene – but also unspecifically for other genes. The reason
is that this is „sensed“ by the cells like a long viral dsRNA, leading to virus
defense mechanisms: activation of PKR (protein kinase R), phosphorylation of
translation factors and general downregulation of protein synthesis.
Some years ago, scientists found that small dsRNA in the range of 19-21
nucleotides interferes specifically with target genes, without affecting other
genes (small interferent RNA, siRNA) – because they are too small to activate
virus defense mechanisms.
66
Principle of RNA-interference
Small dsRNA (siRNA) binds to an
RNA-induced silencing complex
(RISC); consisting of argonaute
proteins.
Together with the RISC, one RNA
strand binds to the target mRNA
and leads to specific degradation
of this mRNA.
The RISC complex is „recycled“
leading to degradation of addition
mRNA-targets.
67
33
micro-RNA‘s – the biological mechanism to
suppress gene expression
miRNAs – can have
two effects:
1) mRNA degradation
2) inhibition of
translation (this
effect often does
not require a
100% match with
the target mRNA!)
Chemically synthesized siRNA
Design: traditionally the first AA-Duplett is searched – and the following 19
bases are checked for GC-content (should be 40 – 50%), the sequence
should be target gene specific (checked by BLAST) – an appropriate RNA
sequence and the reverse complementary RNA are chemically synthesized
(and ordered by a company, e.g. Dharmacon, Invitrogen, MWG,…).
Company homepages often offer a basic siRNA design:
http://www.dharmacon.com/
http://www.mwgbiotech.com/html/s_synthetic_acids/s_rna.shtml
The two short RNAs are annealed and transfected (usually using
methods that are suited for short oligonucleotides (e.g.
Lipofectamine2000 from Invitrogen, XtremeGene from Roche…).
69
34
Vector-coded siRNA > small hairpin RNA (shRNA)
•the normal transfection methods, optimized for plasmids can be used
•Antibiotics selection genes (e.g. G418, puromycin…) can be included > stable
„knock-down“ cell lines can be generated (www.imgenex.com,
http://www.oligoengine.com/)
70
small hairpin RNA: hairpin-loop
71
35
Professional Design of siRNA or shRNA
•
•
•
•
•
Design via company website
http://www.thermoscientificbio.com/design-center/?redirect=true
This delivers a list of several possible sequences (gene specific,
checked by BLAST) – with a score based on empirically determined
criteria: Nature Biotechnology 22, 326-330, 2004
Check literature for functional siRNA sequences
For transduction of primary cells: lentiviral shRNA constructs
(also work in non dividing cells)
there are also inducible lentiviral constructs available
(http://tronolab.epfl.ch/)
Many vectors can also be obtained from plasmid repositories:
Addgene: http://www.addgene.org
Belgian repository: http://bccm.belspo.be/db/lmbp_search_form.php
72
Gene replacement strategy
5‘UTR
gene of interest
3‘UTR
endogenous mRNA
siRNA
targeting the endogenous
mRNA via the
untranslated region
Expression plasmid containing:
good
promoter
foreign
3‘UTR
mutated gene of interest
(SV40
PolyA)
the mutated gene
replaces the endogenous
gene
73
36
Important controls in siRNA experiments
• scrambled siRNA as negative control
• mutated siRNA with some mismatch as negative control (note:
might act as miRNA !)
• other siRNAs targeting the same mRNA should have the same
effect
• if you use shRNA (vector based RNA-interference) use an
unrelated shRNA as negative control (e.g. shRNA vector with
scrambled siRNA sequence).
The empty shRNA vector is not a valid negative control
Alternative Methods to influence endogenous
protein levels
Micro-Injection:
This allows injecting antibodies
against certain endogenous
proteins > interfering with their
functions. However, just a limited
number of cells (e.g. up to
hundreds with automated systems)
can be targeted > the following
analysis should be a single cellbased assay, such as microscopy.
75
37
Research Methods - Overview
•
•
•
•
•
•
•
•
cell culture systems
labelling and transfection of cells
analyses of cellular components
analyses of molecular interactions
fluorescence measurements
microscopy
flow analysis (FACS)
analyses of cellular processes (proliferation, apoptosis..)
76
Analysis of Proteins by SDS-PAGE
S-S
SO4-
reducing agent (DTT)
or Mercapto-Ethanol
- breaks disulfide
bonds
95°C
-
- - - - - - -
SDS – sodium dodecyl
sulfate: coats proteins
with negative charges
- - -
-
-
-
77
38
SDS-Gels
For final concentration of gel ( % T):
Stack gel
Separating gel (10 ml)
30% Acrylamide-bis
solution 29:1
(A)
(10 ml)
7%
10%
12,5 %
15%
5%
2.33
3.33
4.17
5
1.67
2.5
2.5
2.5
2.5
6x SDS-buffer
4x Separation buffer
1.5 M Tris/HCl pH
8.8
2 M Tris-Cl
(pH 6.8)
4x Stacking buffer
0.5 M Tris/HCl pH
6.8 + phenol
red
aqua dest.
2.5
5
4
3.2
2.4
5.7
0.1
0.1
0.1
0.1
0.1
TEMED
0.015
0.015
0.015
0.015
0.015
APS (10%)
0.03
0.03
0.03
0.03
0.03
SDS (10 %)
2.4 ml
SDS
0.96 g
Glycerol
4.8 ml
DTT
739 mg
Bromophenol
Blue
4.8 mg
78
Detection techniques
Coomassie-Blue staining: robust,
moderate sensitivity (limit ≈ 1 µg)
Silver staining: elementary silver is
deposited at the site of proteins,
very sensitive (limit ≈ 10 ng)
protein-specific fluorescent dyes:
SYPRO-Orange, SYPRO-Ruby,
Deep-Purple
(compatible with MALDI-TOF, MS)
Special stainings: Proteoglycans
(Alcian-Blau), glycoproteins
(Schiff‘s Reagent)
Autoradiographie, Fluorography
79
39
Silver staining
66 kDa
25 kDa
separation with 12% Acrylamide: 20 - 80 kDa
80
Molecular weight assessment after SDS-PAGE
http://www.meduniwien.ac.at/user/johannes.schmid/SDS-PAGE.xls
log[MW]
migration distance starting from
stacking gel/separation gel interface
81
40
Fluorography, Autoradiography
Sensitive detection of radioactively labeled proteins
Gel is equilibrated with a radiosensitive fluorophore:
e.g. Diphenyloxazole (POP), Sodiumsalicylate
Detection by X-ray film or Phoshor-Imager devices
82
Fluorography
Phospho-Imager detection
(1 day exposure)
X-ray film detection
(3 months exposure !!)
83
41
Silver Staining of PAGE Gels
Solutions
Fixing solution: 50 % ethanol, 10 % glacial acetic acid, ad 100 % with aqua dest.
Incubating solution (1L): 30 % ethanol, sodiumthiosulfate anhydrous 2g, sodiumacetat
anhydrous 34 g, fill up to 1L with aqua dest. Before use add 125 µL of
glutaraldehyde/50 mL incubating solution.
Silvernitrate solution (1L): AgNO3 1 g, dissolved in 1L aqua dest.. Before use add 10 µL
of formaldehyde/50 mL of silver nitrate solution.
Developing solution (1L): Na2CO3 anhydrous 25 g, dissolved in 1L aqua dest.. Before
use add 10 µL of formaldehyde/50 mL of developing solution.
Stop solution (1L): sodium-EDTA 15.78 g dissolved in 1L aqua dest..
After electrophoresis, the polyacrylamide gel is taken out of the casting sandwich and
placed in a clean glass beaker filled with fixing solution. All following steps are
carried out while gently shaking. The gel has to be incubated with the fixing
solution for 30 minutes. After fixation an appropriate amount of incubating solution
including glutaraldehyde (the gel has to be at least covered by liquid) is prepared
and added to the gel, followed by incubation for 15 minutes, discarding the fixing
solution and washing with aqua dest. 3x for 5 minutes and 10 minutes incubation
in silvernitrate solution including formaldehyde. The silvernitrate solution is
collected (special waste). Developing is carried out by incubating the gel in
developing solution including formaldehyde until the desired intensity of protein
staining is reached, followed by discarding of developing solution and adding stop
solution. The gel should incubate for at least 1 hour in the stop solution.
84
Afterwards the gel can be stored in aqua dest. or dried with vacuum.
EMSA‘s (Electrophoretic mobility shift assays)
…used to monitor active transcription factors (by binding to short, labeled
oligonucleotides comprising the bound DNA sequence)
Example:
comp.: competitor: non-labeled
ds-oligo of the same sequence
(usually added in > 10-fold
molar excess) – competes with
the labeled oligo for binding to
the TF > reduces the specific
signal
mut.comp.: mutated competitor:
should not compete for specific
binding
42
Defining the composition
of TF-complexes using
antibodies and supershifts
supershift
EMSA Alternative: ABCD Assay
(Avidin-Biotin Complex with DNA)
TF
dsOligo
Biotin
Streptavidin
87
43
Isoelectrical Focussing (IEF)
Separation of proteins
according to their isolelectric
point (pH at which they are
not charged)
Usage of immobilized pHgradients (Ampholines)
native IEF or denaturing IEF
(urea) can be done
88
2D-Electrophoresis
for analyses of complex protein mixtures
(→ Proteomics)
combination of IEF (1. dimension) and SDS-PAGE (2.
dimension)
high separating resolution (> 1000 Spots)
Protein pattern databases are available and software
for pattern comparison
identfication of spots by mass spectrometry or by
immunological methods (immunoblotting)
89
44
Principle of 2D-Electrophoresis
90
2D-SDS-PAGE
91
45
2D-DIGE (Difference Gel Electrophoresis)
Protein extracts from
two different samples
are labeled with two
different fluorescent
dyes (e.g. Cy2 and Cy3)
and mixed (usually
together with a pooled
standard mixture
labeled with a 3rd dye,
eg. Cy5).
2D-PAGE is performed,
followed by scanning of
the gel with the 3
wavelengths exciting
the 3 dyes > the results
are compared by
computer analysis
Gel chromatography (size-exclusion chrom.)
• Separation technique that uses porous bead material in a
column to separate macromolecules according to size.
• Wide range of molecular weights that can be separated
• Much larger molecules can be separated than with SDS-PAGE or
native PAGE
93
46
Gel chromatography
larger molecules are eluted first
94
Gel chromatography
Example
0.017
0.19
SERT
Standards
0.015
44 kDa
SERT
0.17
0.15
0.11
158 kDa
0.009
0.09
0.07
0.007
0.05
0.005
0.03
670 kDa
0.003
0.01
110
105
95
100
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
5
-0.01
10
0.001
0
Ex436/Em510
0.13
0.011
Trp-fluorescence
0.013
min
95
47
Spin desalting or buffer exchange based on gel
chromatography
1.
2.
3.
4.
5.
6.
7.
Place spin column in a 1.5 mL microcentrifuge collection tube.
Centrifuge at 1500 x g for 1 minute to remove storage solution.
Add 300 μL of desired final buffer to the resin bed and centrifuge at 1500 x g for 1 minute;
discard flow-through
Place the equilibrated spin column into a new collection tube
Load your protein sample (130 µl)
Centrifuge at 1500 x g for 2 minutes to collect desalted sample.
Protein sample is now desalted & buffer exchanged and ready for use
96
Immunoblotting
Western Blotting: Electrophoretic protein-transfer
from a gel (mostly SDS-PAGE) onto a membrane
Membranes: Nitrocellulose,
Polyvinyl-difluorid (PVDF)
detection via enzyme-coupled antibodies
Enzymes: - Horse Radish Peroxidase (HRP)
- alkaline phosphatase (AP)
97
48
Immunoblotting II
98
Wet Blotting
equipment
99
49
Blotting conditions
100
Semidry blotting (BioRad)
•
•
•
•
•
•
•
wet Millipore Immobilone-P
membrane in MeOH, rinse with
water, then 1 min. in Blotting buffer:
25mM Tris Base
150 mM glycine
10% methanol
(make a 90% stock without MeOH,
which is then added freshly)
filter 0.2 mm !!!
wet 5 3MM filter papers -> anode
then wetted membrane
then gel
then 5 wetted filter papers
wet cathode, close lid quickly
without moving back and forth.
blot 250 mA for a small Bio-Rad gel
(should be 20, increasing to 30 V)
for 30 min.
101
50
Immunoblotting: various substrates
Chromogenic
detection of HRP
Chemiluminescence
-substrate
102
Western Blot Protocol
•
•
•
•
•
Detection:
block 1 hour to O/N in PBS/5% milk at RT
wash 3x PBS/0.1%Tween 20
1st AB in PBS/Tween, 1 h
wash 3x 5 min PBS/0.1%Tween 20
• add 2nd AB in PBS/Tween (e.g., peroxidase-conjugated donkey
anti-rabbit Amersham, at 1:10.000) for 1 hour
• wash 3x 5 min
ECL-system:
• combine equal volumes of soln. A and soln. B (Amersham ECL or
Pierce SuperSignal WestPico)
• add to filter, incubate 1 min. (Amersham), or 5 min (Pierce)
• roll away, cover with saran wrap, expose within 10 min.
• (exposure times can vary between seconds and 20 min.)
103
51
ECL reagent: selfmade version
Advantages: almost no cost; stable/reproducible, stored in frozen aliquots
To 10 mls of 100 mM Tris pH 8.5 (RT), add
50 µl luminol (warm to redissolve)
22 µl of coumaric acid (warm to redissolve)
3 µl of H2O2 (fresh < 6 months)
Pour onto blot for 1 minute and process as normal (10 ml enough for 100
cm2)
Stock luminol: 250 mM 3-aminopthalhydrazide (Fluka #09253); 266
mgs in 6 mls DMSO; store frozen in 60 µl aliquots.
Stock coumaric acid: 90 mM coumaric acid (Sigma C9008): 38 mgs in
2.5 mls DMSO; store frozen in 25 µl aliquots.
STRIPPING of membranes: with 2% SDS, 62.5mM TRIS pH6.8,
100mM Beta-mercaptoethanol for 30 min at 50°C
104
Immunoprecipitation
Antigen
Interaction between antibody and
antigen in solution
Isolation of immuno-complexes by
Protein A- Agarose (Sepharose) – Beads
Protein A = IgG-binding protein of
Staphylococcus aureus
Alternative: Protein G: often used for
mouse monoclonal antibodies, which are not
well bound by Protein A
- or antibody covalently linked to activated
Sepharose (CNBr-activated)
Analysis by SDS-PAGE
105
52
Example of an Immunoprecipitation
Proteins labeled unspecifically with 35S-methionine > specific detection of a
protein of interest by immunoprecipitation > fluorography
106
Affinity chromatography
• beads can be coupled
with antibodies or other
affinity ligands, which
bind a molecule of
interest.
• after washing of the
beads, the captured
target molecules can be
eluted e.g. by lower pH.
107
53
Analysis of post-translational
modifications
proteolytic processing
Glycosylation
Phosphorylation
Ubiquitination
….
108
Analysis of proteolytic processing
in vivo: cultivation of cells in presence of selective
proteinase inhibitors
inhibition of intracellular proteinase (in cytosol, ER,
Golgi) is possible just with membrane permeable
inhibitors
inhibitors that are not membrane permeable act just
in the extracellular environment, in endosomes and
lysosomes
in vitro: Incubation with specific proteinases
109
54
Proteolytic processing (in vivo)
Z-FA-CHN2
Z-FA-CHN2 .... Benzyloxycarbonyl-Phenylalanyl-Alanyl110
Diazomethane (inhibits cysteine proteinases)
Detection of N-Glycosylation
in vivo: Biosynthesis in presence of inhibitors
Tunicamycin: inhibits the initiation of N-Glycosylation
Processing of N-Glycans blocked by GlycosidaseInhibitors
Tunicamycin (µg/ml)
0
0.1 0.5
111
55
Analysis of Glycosylation (in vitro)
Endo H .... Endoglucosaminidase H; cleaves just
Mannose-rich N-Glycans
PNGase ... Peptid:N-Glycosidase F ("N-Glycanase")
112
Analysis of Phosphorylation
in vivo: metabolic labelling with
[32P]Phosphate (e.g. in presence or
absence of an expression or suppression
construct for a specific kinase, followed by
immunoprecipitation)
in vitro:
- Incubation with alkaline Phosphatase
- Phospho-aminoacid analysis (DC)
- Kinase-Assays
Immunoblotting/Immunprecipitation
(e.g. with anti-phospho- specific Antibody
or for instance anti-Phosphotyrosin)
113
56
In vitro Phosphorylation Analysis
PNGase ... Peptid:N-Glykosidase F ("N-Glykanase")
AlkPhos ... Alkaline Phosphatase
114
in vitro Kinase Assay
1.
Immunoprecipitation of the kinase
2.
Kinase-Reaction in presence of
3.
SDS-PAGE and Fluorography
32P-γ-ATP
32P-γATP
+
substrate
phosphorylated substrate
Immunoprecipitated Kinase
antibody
Agarose-Bead
115
57
Kinase Assay- Protocol
Lysis buffer (final conc.):
for 20 ml:
20 mM Tris/HCl pH7.5400 µl 1 M150 mM NaCl600 µl 5 M25 mM b-glycerophosphate500 µl 1 M2 mM EDTA80 µl 0.5 M2 mM
pyrophosphate400 µl 0.1 M1 mM orthovanadate200 µl 0.1 M1% Triton X-1002 ml 10%1 mM DTT20 µl 1 M1 mM
NaF20 µl 1 MA. dest.15.8 ml
Protease Inhibitors: added before use (Leupeptin, Pepstatin, Pefa-Block) according to stock
Kinase buffer (final conc.):
for 20 ml:
20 mM Tris/HCl pH7.5400 µl 1 M20 mM b-glycerophosphate400 µl 1 M100 µM orthovanadate20 µl 0.1 M10 mM
MgCl2200 µl 1 M50 mM NaCl200 µl 5 M1 mM DTT20 µl 1 M50 µM ATP50 µl 20 mM1 mM NaF20 µl 1 MA. dest.18.7
ml
Lyse cells (in 6 wells) with 500 µl per well of Lysis buffer (+ protease inhibitors):
20 min at 4°C.
Clear by centrifugation (14000 rpm, 4°C 15 min Eppendorf centrifuge). Save an aliquot (30 µl) for Western blotting.
Immunoprecipitate the kinase (e.g. with 10 µl anti-flag affinity matrix beads, Sigma, for flag-tagged transfected kinase; or
with appropriate antibody for endogenous kinase + Protein A-Sepharose or directly coupled to agarose): 2h at 4°C
(rotating).
Wash the beads: 3x with 1 ml PBS (4°C), 1x with 1 ml Kinase buffer (4°C): pellet the beads by centrifugation (14000 rpm,
4°C, 45sec) and remove the supernatant.
Prepare Kinase buffer: add MnCl2 to 10 mM (stock: 1 M) and 32P-g-ATP (5 µCi per sample, usually 1/10 volume, i.e. 1 µl
of stock solution for one 10 µl assay) and preincubate at 30°C for 10 min.
Add 1 µg substrate: GST-IkB (1 µl) or mutant substrate (as control) to the beads; add 10 µl kinase buffer, mix gently and
incubate at 30°C for 30 min (or longer).
Stop the reaction by addition of 4x SDS-sample buffer (4 µl) and perform SDS-PAGE with the samples, followed by fixation
of the gel (10% methanol, 10% HAc), drying and autoradiography.
For detection with PhastGel: use only 5 µl beads, 5 µl kinase buffer, 0.5 µl substrate and 2 µl 4x SDS-sample buffer:
Run a
116
12.5% PhastGel with 4 µl per sample
Detection of Ubiquitination
Potential set-up:
•
•
•
•
•
•
transfection of a tagged ubiquitin
(e.g. His-tagged) together with the
gene of interest (e.g. flag-tagged)
immunoprecipitation of the gene
of interest
optional: resuspend and heat the
beads in 1% SDS-buffer, dilute to
0.05% SDS and repeat
immunoprecipitation to get rid of
potential co-precipitating,
interacting proteins.
resuspend and heat the beads in
SDS-PAGE buffer
SDS-PAGE
Western Blot for the His-tag
58
Subcellular Fractionation
(Separation of subcellular compartments)
most often used: density gradient centrifugation
coarse separation: differential centrifugation
Detection of subcellular compartments by specific
markers (enzymes that are nearly exclusively in that
compartment)
118
Differential centrifugation
Subsequent centrifugation steps with increasing g-force
119
59
Gradient centrifugation:
Samples are usually layed on top of a gradient, proteins
or compartments migrate through the gradient in „zones“
120
Density gradient centrifugation
Gradients: continuous ↔ discontinuous („steps")
self-forming gradients (e.g. Percoll)
(density equilibrium centrifugation)
samples are either layered on top of the gradient or at the
bottom
Fractionation after the centrifugation (e.g. by peristatic
pump and fraction collector)
gradient mixer:
B
A
0
100
A
100
Magnetic stirrer
B
0
volume (or time)
121
60
Substances to generate density gradients
Sucrose: low molecular weight (342 Da),
osmotically active
Ficoll: copolymerisate of Sucrose and
Epichlorhydrine; Mr ≈ 400 000 Da
Percoll: colloidal silica gel
special case for DNA: Cesiumchloride
122
Self-forming gradients (e.g. Percoll..)
123
61
Example for a density gradient centrifugation
Separation of lysosomes ( ) and Golgi ( )
in a continuous Percoll gradient (density º)
124
Research Methods - Overview
•
•
•
•
•
•
•
•
cell culture systems
labelling and transfection of cells
analyses of cellular components
analyses of molecular interactions
fluorescence measurements
microscopy
flow analysis (FACS)
analyses of cellular processes (proliferation, apoptosis..)
125
62
Methods to investigate macromolecular interactions
•
•
•
•
•
•
•
Interaction screening with phages (Phage Display)
Yeast 1-Hybrid System (protein : DNA)
Yeast 2-Hybrid System (protein : protein)
Mammalian 2-Hybrid System
Gel-Chromatography
Co-Immunoprecipitation
Fluorescence Resonance Energy Transfer (FRET)
– see fluorescence methods
126
Interaction screening with phages
(Phage Display)
A gene library is expressed on the
surface of appropriate phages (e.g.
M13), which are incubated with specific
target proteins immobilized on plates.
Unbound phages are washed off; bound
phages are eluted by lowering the pH.
3-4 x
Bound phages are amplified and again
incubated with plates containing the target
proteins – this repeated 3 – 4x to enrich the
specifically binding phages. Clones are isolated
and sequenced > Sequence of the binding
protein
127
63
Yeast 1-Hybrid System
For the identification of proteins that bind specifically to a given DNA sequence (e.g.
transcription factors; DNA:protein interaction).
The DNA sequence of interest (e.g. from a promoter) is usually cloned in repeats (3-5x)
in front of an appropriate selection gene (e.g. a histidine synthesis gene) and an
appropriate reporter yeast strain (which is not capable of growing in the absence of
histine) is stably transformed with this construct. Subsequently, this yeast strain is
transformed with a library containing putative binding proteins (often fused to the
transactivation domain of the Gal4 transcription factor). Binding of a protein to the DNA
sequence results in growth of this yeast clone on selection plates.
Gal4-Activation domain
Transcription
Insert from library
TATA
HIS
> growth on
selection plates
lacZ
DNA-Region with potential protein
binding sites (in repeats)
128
Yeast 2-Hybrid System
A yeast strain is used, which does not contain a functional Gal4 transcription factor –
but reporter and selection genes, which are downstream of Gal4-dependent promoters
(Histidine- and Adenine-synthesis genes, lacZ for β-Galactosidase expression, which
can be used for staining). This strain is transformed with putative interaction partners:
1. fusion protein of the Gal4-DNA-binding
domain and Protein X („bait“)
2. fusion protein of the Gal4transactivation domain and Protein Y (or a
library insert; = „prey“)
Co-Transformation or combination by
yeast mating
> In case of an interaction between protein X
and Y auftritt, a functional transcription factor is
build, which binds to Gal4 promoters – and the
cells can grow on selection plates (without His
or Ade).
129
64
Preparation of a Yeast 2-Hybrid Screen
1.
Cloning of the gene of interest into the bait
vector“ (in frame with the Gal4-DNAbinding domain): selection in bacteria (e.g.
via Kanamycin), selection in yeast (e.g. via
Trp-synthesis gene))
2.
Test for Auto-Activation with „empty
Gal4AD-vector): Tests whether the gene of
interest interacts with the Gal4 activation
domain (without the need of protein Y):
If it does so, the bait cannot be used in the
yeast 2-hybrid system.
The Gal4 AD vector contains a second
selection gene for yeast (e.g. leucine
synthesis gene). Co-transformants of the
two vectors grow in the absence of Trp and
Leu; but they should not grow in the
absence of the amino acid that is
synthesized just when an interaction occurs
(e.g. in the absence of histidine or
130
adenine).
Combination of the two putative interaction partners
Reporter strain Y187
pretransformed with a library (in
Gal4AD-Vector), Mating Type: α
Reporter strain
(e.g. AH109)
Mating Type: a
bait
X
Gal4AD
Gal4BD
Mating: Incubation of the two haploid
strains for 24 h at 30°C, 40 rpm
> formation of diploid clones with both
vectors
clones, which contain interaction
partners grow on selection plates and
express lacZ
Instead of mating the two vectors can be
combined by classical transformation
X
His, Ade,
lacZ
PCR from single colonies (with primers specific for the library vector)
purification of PCR-Products
sequencing of the putative
interaction partners
131
65
Example of a yeast two-hybrid result
A) single colonies on selection
plates (SD-Leu-Trp-Ade)
B) Streaking out the colonies from the
first selection plates to secondary
selection plates (e.g. with higher
selection pressure and stringency:
SD-Leu-Trp-Ade-His)
132
Verification of a yeast 2-hybrid result
1.
Analysis of the sequence and comparison with database: check whether the ORF
is OK (in frame with the Gal4AD)
2.
Isolation of Plasmid-DNA from the yeast colony
3.
Re-transformation in E.coli (to separate bait and prey – using different antibiotics
resistance) and preparation of the plasmid containing the library insert
4.
„False Positive Test“ in yeast: Transformation of the Gal4AD-plasmid containing
the identified „prey“ with the empty Gal4-binding domain vector: this shouzld not
lead to growth on selection plates of interaction (if there is growth, then the
library insert interacts with the Gal4BD and not the bait protein)
5.
β-Galactosidase-assays (also quantitative, to compare interaction partners)
6.
Verification in the correct cells (human cells), e.g. by co-immunoprecipitation
133
66
Example of a “False-Positive Test”
1,7
1,6
rel. activity
1,5
Example of lacZ
quantification
1,4
1,3
1,2
1,1
1
neg. control
IKK2/GMRa
IKK2/GMRb
134
LiAc Yeast Transformation
•
•
•
•
•
•
•
•
Solutions:
Synthetic drop out solution 10 x in AD: L-isoleucine 300 mg/L, L-valine 1.5 g/L, L-adenine hemisulfate salt 200
mg/L, L-arginine HCl 200 mg/L, L-histidin HCl monohydrate 200 mg/L, L-leucine 1 g/L, L-lysine HCl 300 mg/L, Lmethionine 200 mg/L, L-phenylalanine 500 mg/L, L-threonine 2 g/L, L-tryptophan 200, L-tyrosine 300 mg/L, Luracil 200 mg/L
SD -Trp medium (synthetic dropout medium): synthetic minimal medium lacking tryptophan: yeast nitrogen
base without amino acids 6.7 g/L, 2 % dextrose (glucose) (sterile dextrose solution is added after autoclaving
to avoid maillard reactions), pH adjusted to 5.8, for plates : agar 1.5 g/L
YPD (yeast peptone dextrose) broth, yeast complete medium: yeast extract 10 g/L, peptone 20 g/L, 2 %
dextrose (glucose), pH adjusted to 5.8
DNA + water + carrier-DNA or RNA
Aqua dest. sterile
1M LiAc
LiAc 100 mM sterile
Polyethylenglycol
LiAc 1 M sterile
Bacterial RNA, used as carrier
yeast pellet
Poly-ethyleneglycol PEG 50 % (w/v) sterile filtered
10 mL of SD -Trp medium are inoculated with the appropriate yeast strain and incubated at 30°C while shaking at 200
rpm o/n. On the next day OD at 600 nm is measured and the yeast culture is diluted with YPD to OD600 0.1. A
total volume of 50 mL diluted yeast culture is used for further incubation. Every hour OD600 is measured until
OD600 0.4 is reached (3 - 5 hours). Then the cell number is calculated with a Thoma chamber. 2x107 cells/mL
are sufficient for 10 transformations. The yeast is then harvested by centrifugation at 3000 rpm for 5 minutes,
the supernatant is carefully removed and collected for autoclaving. The pellet is resuspended in 25 mL sterile
AD and again centrifuged at 3000 rpm for 5 minutes. After removing of the supernatant the pellet is
resuspended in 1 mL LiAc 100 mM. Excess of LiAc is removed by spinning the tubes for 15 seconds at full speed
in a tabletop centrifuge and carefully removing the supernatant. The yeast pellet is brought to a final volume of
500 µL with LiAc 100 mM. Aliquots of 50 µL are prepared. One 50 µL aliquot of this yeast suspension is used for
one transformation. 50 µL aliquots are again briefly centrifuged to pellet the cells, the supernatant is removed
and on top of the yeast pellet, layers of the following transformation solutions are pipetted in following order:
240 µL 50 % PEG, 36 µL LiAc 1 M, 3.3 µL of bacterial RNA (31 µg/µL), 70.7 µL sterile AD, 1 µg plasmid DNA
(1µg/µL). The tube is then thoroughly mixed by vortexing for 1 minute until the yeast pellet is completely
dissolved and placed for 30 minutes in a 30°C water bath. The tube is then transferred to a 42°C water bath for
25 minutes in order to perform the heatshock. The transformation mix is then briefly centrifuged for 15 seconds
at 4 000 x g (7 000 rpm) in a table top centrifuge, the supernatant is discarded and the pellet is resuspended
135in
1 mL sterile AD. 50 µL of this transformed yeast suspension are plated on SD – Leu, - Trp, - Ade plates and
incubated at 30°C for some days.
67
Mammalian 2-Hybrid System
Posttranslational modifications such as
phosphorylations, which might be
essential for interactions are often not
carried out in yeast. In this case a
similar assay can be set up in
mammalian cells (e.g. providing the
kinase)
Limitations:
1.
not suited for screening
purposes
2.
Proteins are in the nucleus
and thus eventually not at
their normal localization
> FRET-Microscopy: as alternative to
visualize protein-protein-interaktion in
their physiological context
136
Example for a mammalian 2-Hybrid Test
137
68
Biochemical Verification of Protein-Interactions
by Co-Immunoprecipitation (CoIP)
1.
The 2 proteins of interest are transfected into mammalian cells (usually containing
to different tags, e.g. HA- and flag). 1 – 2 d after transfection, the cells are lysed
and one protein is immunoprecipitated using antibody-beads against tag1 (e.g.
flag). The beads are washed extensively with buffer (isotonic or hypertonic, not
hypotonic) and finally heated with SDS-buffer to release bound proteins. SDSPAGE and Western blotting is performed – using antibodies against tag1 and
against tag2. If protein with tag2 co-precipitated with protein containing tag1, then
there is interaction.
2.
Co-immunoprecipitation of endogenous proteins (without transfection) – using the
same principle and antibodies against the endogenous proteins
1.
Protein
X-myx
Protein
Y-HA
3.
2.
HRP
Antikörper
bead
138
Example for a Co-Immunoprecipitation
1. Co-IP with overexpressed proteins
(after transfection); control:
transfection with just one protein
2. Co-IP with endogenous proteins
control: IP with unrelated antibody
139
69
The salt concentration has an influence on the
stringency of the co-immunoprecipitation
Co-Immunoprecipitation of TRAF1 and
TRAF2 at 500 mM NaCl
Co-Immunoprecipitation of TRAF1 und
IKK2 works at 250 mM NaCl, but not at
500 mM > TRAF1/TRAF2 interaction is
stronger than TRAF1/IKK2 interaction
140
Co-Immunoprecipitation for the Detection of Protein Interactions
1. Transfection of cells with tagged proteins (one 6-well of CHO or HeLa cells is sufficient for one sample).
2.
Preparation of extracts:
2.1. 1 d after transfection: wash cells with PBS
2.2. Lysis with 500 µl/well Lysis-Buffer + Protease Inhibitors: 15 min at 4°C.
Buffer: 0.5% NP40, 50 mM Tris/HCl pH 7.5, 1 mM EDTA, 150 mM NaCl.
Protease Inhibitors: 10 µg/ml Aprotinin, 20 µg/ml Phosphoramidon, 40 µg/ml Pefabloc, 1 µg/ml Leupeptin, 1 µg/ml
Pepstatin (from 1000x stock solutions, Boehringer Protease Inhibitor set).The lysis is suited for cytosolic proteins and
membrane proteins. Nuclei remain intact (you can leave the nuclei on the plate when you take off the supernatant).
2.3. Spin the extracts for 15 – 30 min at 14 krpm, 4°C (HeLas: 15 min, CHO: 30 min)
2.4. Keep the supernatant and adjust the NaCl-concentration (150 mM – 1000 mM depending on the strength of
interaction; start in the range of 150 – 250 mM, increase the concentration if you want to increase the stringency)
3.
Co-Immunoprecipitation
3.1. Take 400 µl of extract for IP (keep about 30 µl extract for direct western analysis).
Use flat-top tubes (the visibility of the pellet is better in these tubes) Add 400 µl Lysis-Buffer/250 mM NaCl (without
NP40 > final concentration: 0.25%). Add beads (15 µl anti-flag-M2-Agarose, Sigma A-1205; alternatives: other
antibodies directly coupled to CNBr-activated Sepharose; Protein A- or Protein G-Agarose: the later will give more
unspecific binding). Rotate extracts + beads for 2 h at 4°C.
3.2. Spin for 30 sec at 14 krpm 4°C. Take off the supernatant, add 1 ml of lysis buffer/250 mM NaCl/without NP40
and invert tubes several times (do not vortex). Repeat this washing step.
3.3. Suspend the beads in 1 ml cold PBS and transfer the suspension to a new tube.
Spin 30 sec at 14 krpm, 4°C, take off the supernatant and repeat this washing step. Final centrifugation: 1 min at 14
krpm, 4°C. Remove the supernatant and suspend the beads in SDS-PAGE buffer (30 µl). Incubate for 5 min at 95°C
and pellet the beads for 2 min at 14 krpm.
4.
SDS-PAGE
5. Western Blot: if possible use HRP-conjugated primary antibodies (anti-HA-HRP from Boehringer, anti-mycHRP from Invitrogen). This gives much lower background of unspecific bands (Ig light chain …).
141
70
Far Western Blotting
… the membrane is probed with a protein, which can bind the protein of
interest. While western blotting detects certain proteins using antibodies, farwestern blotting detects protein:protein interactions.
Research Methods - Overview
•
•
•
•
•
•
•
•
cell culture systems
labelling and transfection of cells
analyses of cellular components
analyses of molecular interactions
fluorescence measurements
microscopy
flow analysis (FACS)
analyses of cellular processes (proliferation, apoptosis..)
143
71
Principle of Fluorescence
1. electrons of a fluorophore are
excited by absorption of an
appropriate photon (hν −Ex) and
their energy state is raised to S1´
Jablonski-Graph
2. the excitated state S1´exists for
about 1 – 10 nsec. Energy is lost
by several reactions
(interaction…) leading to the
excited state S1.
3. Electrons fall back from S1 to S0 – the energy difference is
released by emission of a photon (which has lower energy than the
excitation photon – and thus a longer wavelength) according to
λ = c/ν
144
Principles of Fluorescence
double bonds = flexible (delocalized) p-electron system
from:
http://www.invitrogen.com/site/us/en/home/support/Tutorials.html
72
delocalized p-electron systems
(alternating double bonds) can
easily absorb photons and
thereby be raised to higher
energy levels
delocalized p-electron systems
(alternating double bonds) can
easily absorb photons and
thereby be raised to higher
energy levels
73
energy loss due to
movements, rotations etc...
... sudden fall from an excited
energy level to the ground
state
74
The absorbance of light
(photons) depends on the
colour (the wavelength)
number of
absorbed
photons
excitation spectrum
75
The emitted light (photons)
exhibits a certain wavelength
spectrum (colour) –
depending on the nature of
the fluorophore
number and colour of
emitted photons
emission spectrum
153
76
Excitation and Emission Spectra
Stoke‘s Shift
Infos: http://www.probes.com/servlets/spectra/
Java-Applet from BD: http://www.bdbiosciences.com/spectra/
154
Java-Applet from BD:
http://www.bdbiosciences.com/spectra/
155
77
Characteristics of fluorescent dyes
Excitation Maximum: wavelength of maximal photon
absorbance (λ in nm)
Emission Maximum: wavelength of maximal photon
emission (fluorescence, λ in nm)
Molar Extinction coefficient: gives the absorbance of
excitation photons at the excitation maximum λ (in cm1M-1)
Quantum-Yield: number of emitted photons per
number of absorbed photons.
Brightness = molar extinction coeff. x quantum yield
156
Parameters of some important fluorescent dyes
dye
Ex
Em
DAPI
359
461
FITC
494
520
TMRho
550
573
TexasRed
595
615
DAPI... 4’,6-Diamidino-2-Phenylindol
FITC... Fluorescein Isothiocyanat
TMRho...Tetramethylrhodamine
(TRITC: Tetramethylrhodamine Isothiocyanate)
157
78
Alexa-Fluorophores from Molecular Probes/Invitrogen
(www.invitrogen.com)
158
GFP (Green Fluorescent Protein) and its variants
•
Structure: barrel like with the chromophore in
the middle
•
MW: appox. 29 kDa
•
original protein from jellyfish (Aequorea
victoria), exists in bacterial and mammalian
codon optimized versions.
•
Point mutations were incorporated improving
the fluorescence (enhanced GFP: EGFP) and
also leading to other spectral variants (colours;
ECFP, EYFP…)
•
Fluorescent in living cells – can be expressed
as fusion protein with the protein of interest (is
usually not altering the function of the target
protein)
159
79
Fluorescence Properties of some GFP-Variants
Variant
Excitation (nm)
Emission (nm)
EBFP (Blue)
380
440
ECFP (Cyan)
433
475
EGFP (Green)
488
507
EYFP (Yellow)
513
527
DsRed
558
583
160
Other fluorescent proteins
fluor. protein
Ex-Peak
nm
Em-Peak
nm
quantum yield
comment
EBFP
380
440
0.18
ECFP
433 (453)
475 (501)
0.4
Clontech
Clontech
EGFP
488
507
0.6
Clontech
397 (475)
509
0.77
Aequorea victoria
EYFP
513
527
0.61
Clontech
Citrine
516
529
0.76
Griesbeck et al. 2001
DsRed
558
583
0.29
Clontech, tetramer
0.55
tetramer,
wildtype GFP
DsRed2
563
582
588
618
0.02
Clontech, dimer
PA-GFP
(Patterson
2002)
400 before act.
504 (397)
after
515 before act.
517 after act.
0.13
0.79
photoactivatable GFP, T203H
mutant of mammalian
codon-optimized wildtype
GFP
PS-CFP
400 before act.
490 after act.
468 before act.
511 after act.
0.2
0.23
photoswitchable CFP, turns from
cyan to green after intense
illum. at 405 nm
mOrange
548
562
0.69
Shaner et al., 2004
596
0.29
Shaner et al., 2004
610
0.22
Shaner et al., 2004
581
0.69
Shaner et al., 2004, dimeric
HcRed1
mStrawberry
mCherry
dTomato
574
587
554
161
80
Photoconvertible fluorescent proteins
mOrange conversion to far-red
(2x bleaching with 100% 488 nm in between)
120
100
rel fluor
80
mOrange
60
converted far red
40
control cell
20
0
0
20
40
60
80
100
120
sec
mOrange
Far-Red
Photo-switchable fluor. protein Dronpa
120
start bleaching at 488 nm
% of initial fluor.
100
80
60
start reactivation
at 350 nm
40
20
0
-20
0
20
40
60
80 100 120 140 160 180 200
sec
81
Fluorimetric Analysis Methods
Fluorescence measurements are usually more sensitive than
photometric measurements.
Scanning fluorometers have usually 2 monochromators for adjusting
excitation and emission wavelengths. Most instruments also allow to
adjust the bandwidth of excitation and emission (between 1–20 nm)
The emitted fluorescence is measured by a photomultiplier tube
(PMT) detector. The sensitivity of that can be adjusted by changing
the voltage (e.g. 400 - 700 V).
Emission
Monochromators
Detector
164
Parameters of fluorometry
•
•
•
•
•
•
Excitation wavelength in nm
bandwidth of the excitation light (1 – 20 nm, „slit width“)
Emission wavelength in nm
bandwidth of the emission
sensitivity of the detector („gain“, voltage of the PMT)
Integration time of the measurement (slow – fast, in sec.:
influences the „noise“)
165
82
Wavelength Scans
The exact emission and
excitation peaks might differ
slightly between different
fluorometers.
Emission spectrum (ECFP)
2
For checking the parameters:
- run an excitation wavelength
scan at the literature value of the
peak emission
- run an emission scan at the
determined excitation peak
- repeat the excitation scan at
the determined emission peak
rel. fluor.
1.5
1
0.5
0
460
480
500
520
540
nm
560
580
For adjusting these parameters
you have to consider the
fluorescence properties (e.g. the
Stoke‘s shift) – to prevent that
excitation light is detected
166
Tricks for optimizing fluorescence
measurements based on the spectra
excitation
window
possible emission
window
Fluor.
real
emission
curve 1
theor.
excitation
curve
spill-over of the excitation light
theor.
emission
curve
detected fluorescence
nm
83
Tricks for optimizing fluorescence
measurements based on the spectra
Fluor.
excitation
window
more
narrow
emission window
theor.
excitation
curve
emission curve 2
detected fluorescence
nm
Tricks for optimizing fluorescence
measurements based on the spectra
left shifted
broader excitation
window
Fluor.
real
emission
curve 1
real emission curve 3
detected fluorescence
nm
84
Time Scans
Can be applied to determine the time course of fluorescence changes (e.g. to
determine enzyme reaction kinetics – or for instance in chromatography to
measure the kinetics of elution and thus the molecular weight of a fluorescent
compound, such as a GFP-fusion protein)
0.017
0.19
SERT
Standards
0.015
44 kDa
SERT
0.17
0.15
Ex436/Em510
0.13
0.011
0.11
158 kDa
0.009
0.09
0.07
0.007
Trp-fluorescence
0.013
0.05
0.005
0.03
670 kDa
0.003
0.01
110
105
95
100
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
5
10
-0.01
0
0.001
170
min
Fast Kinetic-Analysis
(Stopped-Flow Fluorometry)
Two reaction partners are injected into a mixing chamber, where they are mixed
within approx. 1 msec by stopping the flow.
If the reaction between the two compounds changes the fluorescence, this change
can be recorded with a resolution in the microsecond range.
Light-Absorbance
(Stopped Flow Photometry)
A
Fluorescence
(Stopped Flow Fluorometry)
B
Excitation light
171
85
Example for a stopped-flow fluorometry
0.008
0.007
0.006
0.005
0.004
0.003
0.002
0.001
0.000
-0.001
-0.002
Linear correlation between the initial
reaction kinetics range and the
concentration of the reaction
partners
Tet-DNA
mutant Tet-DNA
0
0.02
0.04
seconds
0.06
rate (1/sec)
rel. fluorescence
Tangent of the initial fluorescence change (reaction kinetics rate in 1/sec)
kon
koff
y = 734,21x + 244,09
R2 = 0,9508
900
800
700
600
500
400
300
200
100
0
rate (15°C)
rate (37°C)
kon
koff
0
0,2
Kaff = 1/Kdiss = kon / koff
0,4
0,6
0,8
DNA (µM)
172
Quantitative Fluorometry
Fluorescence measurements can be used to quantify a great variety of
different substances.
Usually a standard curve is measured with the optimized measurement
parameters (e.g. after defining them by wavelength scans: excitation
and emission wavelengths and corresponding bandwidths; PMT voltage
and integration time).
Some examples for quantitative fluorometry
1.
Enzyme-Measurements (e.g. β-Galactosidase)
2.
DNA-Measurements (Hoechst 33258, SYBR Green): fluorescent
dyes, which intercalate into the DNA and are fluorescent
dependent on the amount of DNA
3.
Protein-Measurements (inherent fluorescence due to aromatic
amino acids such as Tryptophane (dye: SYBR Orange, …)
173
86
Example for quantitative fluorimetric
measurement
Fluorescence (Em 376/ Ex 276)
7
Standard curve
6
Probe
5
4
3
2
y = 0.8738x + 0.1417
2
R = 0.9959
1
0
0
1
2
3
4
5
6
7
8
µg/ml
174
Fluorescence as measurement value in special
analysis techniques: Real-Time PCR
Conventional PCR:
Comparison of a gene of
interest with a housekeeping
gene using an endpoint
determination
Sample 1
In real: samples contain different
amounts of cDNA, but this difference
is not detected, when they reach the
same plateau at the end of the
reaction > this can be revealed by
measuring the reaction product (by
fluorescence) after each cycle
2
1
endpoint
2
175
87
PCR Principle
176
177
88
178
Scheme of a qPCR machine
LightCyclerTM, Roche
heating
Ventilator
capillary with
the PCR-mix
dichroic mirrors
threshold
Ct
light source
detectors
179
89
Realtime PCR machines
Applied Biosystems
Roche Light Cycler
StepOne Plus
capillaries
(app. 1 €/sample)
96-well plates
180
Real-Time PCR with SYBR Green as DNAfluorescence dye
SYBR Green intercalates in the
amplified dsDNA (PCR-product)leading to an increase in fluorescence
with increasing cycle number.
After many cycles the fluorescence
also increases in the water control –
due to the formation of primer
aggregates
181
90
Melting point analysis of the PCR-product
Specific and unspecific PCR
products can be distinguished
by their different melting
temperature (as determined
after the PCR by slow heating
and the decrease of the
fluorescence at the melting
point). This can also be applied
to detect mutations.
measurement of the fluor. after each cycle at a
temperature above the melting temperature of the
unspecific PCR product allows quantifying just the
specific product
182
Real-Time PCR with FRET-Hybridisation Probes
Within the sequence flanked by Primer 1 and
Primer 2 (amplification primers), two additional
oligonucleotides (Hybridisation Probes 1 and 2)
are situated, which contain two different
fluorophores at the 3‘ and 5‘ ends. When these
oligos bind to the PCR product, the fluorophores
come into close proximity and one fluorophore
can transfer part of its fluorescence energy to the
other one (fluorescence resonance energy
transfer, FRET), which then starts to shine. In
this case primer aggregates do not generate a
fluorescence signal.
183
91
Real-Time PCR with TaqMan probes
A TaqMan-probe contains FRET-Donor and Acceptor (Quencher)-Fluorophore within the
same oligonucleotide. At the annealing temperature of the oligo, the probe binds to the PCR
product. The exonuclease activity of the Taq-Polymerase cleaves the probe and leads to
increase of the Donor-fluorescence due to de-quenching.
184
Realtime PCR – Quantification of gene
up/down-regulation
PCR efficiency
CP
1. Determine the PCR efficiency of your
gene of interest and that of your
housekeeping (reference) gene using
serial dilutions (e.g. of plasmids or
cDNA preparations): E = ideally 2
(duplication at each cycle) but
realistically lower (e.g. 1.8)
35
33
31
29
27
25
23
21
19
17
15
y = -1.67ln(x) + 25.767
R² = 0.9972
crossing point CP
Log. (crossing point
CP)
0.1
2. Calculate up- or downregulation of
your specific gene of interest using
the differences in the crossing point
(CP) values with the equation:
ratio =
( Et arg et )
( Eref )
1
10
100
ng cDNA input
∆∆Ct-method
(Pfaffl MW: A new mathematical model for
relative quantification in real-time RT-PCR.
Nucl Acids Res 2001, 29(9):e45
∆CPt arg et ( control − sample )
∆CPref ( control − sample )
(Excel template on my website)
92
100000
Calculating the PCR
efficiency from the
shape of the curve
log (Fluor)
10000
1000
100
- Without using a dilution curve
10
0
10
20
30
40
50
cycle
- Can be calculated for each
sample separately
80000
70000
Neurosci Lett. 2003 Mar 13;339(1):62-6.
60000
Assumption-free analysis of quantitative
real-time polymerase chain reaction (PCR)
data.
Fluor
50000
40000
Ramakers C, Ruijter JM, Deprez RH,
Moorman AF.
30000
20000
10000
0
0
-10000
10
20
30
40
50
(Excel template on my website)
186
cycle
LinRegPCR Software
http://www.hartfaalcentrum.nl/index.php?main=files&sub=LinRegPCR
187
93
188
Research Methods - Overview
•
•
•
•
•
•
•
•
cell culture systems
labelling and transfection of cells
analyses of cellular components
analyses of molecular interactions
fluorescence measurements
microscopy
flow analysis (FACS)
analyses of cellular processes (proliferation, apoptosis..)
189
94
Microscopy: Human vision and the concept of
magnification
image formation in the human eye
2-step magnification principle of a
microscope with 2 lenses:
objective and eye piece (occular)
190
Basics of optical resolution I
Fine structures induce a diffraction of light (light
of zero-order, 1st order ...). Light diffraction on a
small iris is more or less equal to diffraction on
small cellular structures:
sinθ(1) = 1.22(λ/d)
θ ... angle to the first light minimum
λ... wavelength
d ... diameter of the iris
for very small angles θ: θ(1) ≅ 1.22(λ/d)
objects that are closer than θ(1) cannot be resolved as
separate objects
191
95
Basics of optical resolution II
The more orders of light are resolved the better is the resolution.
The optical resolution that can be achieved is defined by the so called
numerical Aperture (N.A.) of the objective.
N.A. = i sin q
i ... Refraction index of the medium
(e.g. 1.0 for air, up to 1.56 for oil)
q... half of the objective opening angle
(Aperture)
192
„Airy“ disks: optical basic structures
193
96
Deceleration (phase shift) of light by passing
through an object
http://www.microscopyu.com/tutorials/java/phasecontrast/phasespecimens/index.html
194
Köhler Illumination
… was established to guarantee optimal
illumination of objects.
This illumination is usually also a
prerequisite for different contrast methods
(phase contrast, differential interference
contrast) to work, as the necessary
components are optimized for Köhler
illumination.
In order to get a Köhler illumination, you
have to focus the object first, then you
close the field iris, so that just the middle
part of the view field is illuminated (if
necessary you have to center the light
path) and then the vertical position of the
condensor is adjusted so that the borders
of the field iris appear sharp and focussed.
195
97
Correct and wrong illumination
field iris at wrong vertical position of the condensor
light path not centered
Correct Koehler illumination
196
Specifications of objectives
•
•
•
•
•
•
Magnification (10x, 20x, 40x…): total
magnification is given by objective
magnification and occular magnification (or
magnification of the lens in front of the CCD
camera)
Immersion medium: air, oil – or water
High magnification (40x – 100x) with oil or
water
additional features (e.g. suitability for
fluorescence due to low autofluorescence of
the glass: Neofluar..)
contrast features: e.g. phase contrast (e.g.
Ph1)
correction of lenses for chromatic
abberations (e.g. Apochromat)
correction of lenses for planarity of focus
over the view field (Plan)
197
98
Contrast enhancement in transmitted light
microscopy
1. Staining of structures
(e.g. nucleus: blue with
hematoxylin, antigen:
brown with immunohistochemistry)
2. Phase contrast:
Making use of the
phase of light when
it passes an object
3. Differential-Interference contrast
(Normarski): Making use of light
polarization and its change through
objects to generate a contrast
198
Phase contrast
Unstained objects such as cells
slow down the light (the phase of
the passing light) by ¼ λ. Phase
contrast rings in the objective can
accelerate the light, which does
not pass through cells by ¼ λ, the
resulting difference of ½ λ causes
an interference, which leads to
contrast enhancement.
¼λ
½λ
http://www.microscopyu.com/tutorials/java/phasecontrast/opticaltrain/index.html
199
99
Phase contrast II
The phase contrast rings of the objektive and the condensor have to match each other in
diameter and have to be concentric. In addition the distance between them has to be
correct (which is the case at the Köhler illumination) – this is especially important for
higher magnification objectives. It is stated on the objektive which phase contrast ring has
to put in at the condensor (e.g. Ph1, Ph2...).
200
phase contrast III
wrong phase contrast ring
correct Koehler illumination and
phase contrast
phase contrast rings not centered
201
http://www.microscopyu.com/tutorials/java/phasecontrast/microscopealignment/index.html
100
Illumination scheme of an inverted microscope
field iris
condensor with phase
contrast rings
Knob to adjust the
vertical position of the
condensor
screws for centering the
light path
202
Differential-Interference-Contrast (DIC)
(Normaski Contrast)
Before reaching the condensor, the light is polarized and
passes a double prism (Wollaston Prism), where it is split
into two beams with different directions and
perpendicular waves. These beams pass the sample,
where they are altered in intensity and phase etc. The
beams are focussed by the objective. In the focal plane
there is a second double prism, which combines the
beams again. After that, the beams are depolarized again.
Thereby the beams that have been altered differentially in
the sample can interfere with each other – and this
interference results in changes of the intensity and the
colour.
The outcome is a preudo-threedimensional image.
203
101
Comparison
Phase Contrast
–
Differential Interference
HeLa cells – same view field
http://www.microscopyu.com/tutorials/java/phasedicmorph/index.html
204
Fluorescence Microscopy
Example: TripleFluorescence-labeled
endothelial cells:
Red: Actin-Filaments
labeled with Phalloidin
Green: Membranes (DiO-C6)
Blue: Nuclei (DAPI-staining
of DNA)
205
102
Basics of fluorescence microscopy
Fluorescent samples are
excited with light of an
appropriate wavelength
(through the objective), the
emitted fluorescence is
collected again by the
objective and is guided to a
dichroic mirror, which
separates the excitation light
from the emitted
fluorescence; the latter
passes an emission filter and
is detected (by eye or by
appropriate detectors such as
cameras)
Interactive Zeiss-Tutorials: http://zeiss-campus.magnet.fsu.edu/tutorials/
206
Scheme of a fluorescence microscope
207
103
Light sources for fluorescence excitation
1.
Conventional light sources:
- mercury lamps:
- Xenon-lamps:
208
LED Light Sources
(light-emitting diodes: semiconductor devices)
from: http://zeiss-campus.magnet.fsu.edu/
104
Metal Halide Lamps
from: http://zeiss-campus.magnet.fsu.edu/
Laser light sources
2. Laser (Light Amplification by
Stimulated Emission of Radiation):
Give just discrete wavelengths
(lines) – thus the choice of
excitation light is limited and
depends on the laser type.
Ar-laser: main lines at 488 nm and
514 nm, (and 458)
He/Neon: 543 nm, 633 nm
UV-Laser: 405 nm
Violet laser diodes: 405 – 420 nm
Advantages of laser light:
-high quality (parallel light beams)
-good for scanning
-high intensity
211
105
Fluorescence Filter Cubes
The filter cube consists of:
1. Excitation filter: just the
correct excitation light
(wavelength) passes the filter
2. Dichroic mirror: is reflective
for the excitation light but
transmittent for the emission
light (the emitted fluorescence)
– separates excitation from
fluorescence light
sample
3. Emission filter: filters the
emitted light so that just the
correct wavelength (e.g. in
double fluorescence) reaches
the detector
212
Characteristics of fluorescence filter sets
Excitation filter
(Band Pass)
Emission filter
(Band Pass)
dichroic
mirror
excitation filter
emission filter
213
106
Example of a bandpass filter + nomenclature
214
Dualband filter sets: Simultaneous observation
of two different fluorophores (e.g. EGFP/DsRed)
Excitation
dichroic mirror
Emission
215
107
Monochromators as light source
A conventional light source (e.g. a Xenon lamp) is split by a
monochromator (e.g. a diffraction grid) to the spectral colours –
to produce light of a freely definable wavelength (320 – 700 nm).
This can be used instead of a fixed excitation filter.
One advantage is that this technology allows switching between
different excitation wavelengths within few milliseconds. This can
be important for excitation ratio imaging (e.g. Fura-2 Calcium
imaging etc.)
UV (320 nm)
electronically
adjustable grid
Polychrome V from
TILL Photonics
Red (700 nm)
216
Detection of the emitted fluorescence
- Visually via the occular of the microscope
- by a CCD camera (usually cooled to reduce the electronic noise).
The photons of the fluorescence hit a light sensitive chip (e.g. out
of 1300 x 1030 pixels), where electrons are released dependent
on the intensity of the fluorescence. Each chip can resolve a given
intensity range – e.g. 256 grey values for a 8-bit camera or 64000
grey values for a 16-bit camera.
The images can be shown on a computer monitor and saved on a
computer
- by photomultiplier tubes (PMT‘s): used often for scanning
devices such as confocal laser scanning microscopes. The gain
(voltage) of the PMT defines the sensitivity (electrons released for
a given number of photons that hit the detector). Often more
„noisy“ than CCD camera images. Averaging is used to smooth
the images (good images takes about 4 sec – while CCD require
just about 100 msec).
- old fashioned: film camera and sensitive film (e.g. 1600 ASA)
217
108
Example for a fluorescence microscopy
experiment
- Cells transfected with fluorescent fusion proteins of a transcription
factor and its inhibitor (appear in the cytosol);
- addition of leptomycin B (LMB) to block nuclear export. This leads to
accumulation in the nucleus indicating continuous nucleo-cytoplasmic
shuttling
218
Example for a fluorescence microscopy
experiment II
Fluorescence was quantified in the nucleus and in the cytosol of the same cell
after different time points > shows the kinetics of nuclear accumulation by the
change of the cytosolic/nuclear ratio.
cytosolic/nuclear fluor.
The data were fitted by nonlinear regression analysis (single exp. decay) –
leading to the half time of the nuclear import process.
5
4
3
2
1
0
0
20
40
min
60
80
219
109
Protocol of an immunofluorescence staining
•
•
•
•
•
•
•
•
Fixation: 15 min 4% Paraformaldehyd
3x 5 min mit TBST wash (50mM TrisHCl pH7.4, 150 mM NaCl, 0.1%Triton)
Block: 1 h at RT with 3% BSA in TBS
Incubation with 1. Ab: anti-IκB (rabbit
polyclonal, sc-371 Santa Cruz)
1:300 in TBS/3% BSA, over night at
4°C (or 1 h at 37°C).
2x 5 min wash with TBST, 1x 5 min
with TBS
Incubation with Alexa488 goat antirabbit 1:2000 in TBS/BSA: 1 h at 37°C
3x 5 min wash with TBST, 1x 5 min
with TBS
Mounting
220
Combinations of transmitted light and fluorescence
IP-Lab Software
A) direct
acquisition with
both light sources
ImageJ software
B) Separate acquisition of fluorescence and phase contrast and merge
or blending (e.g. with ImageJ or other software)
221
110
Interactive Microscopy Demonstrations
Very recommendable:
http://micro.magnet.fsu.edu/
1.
Optical resolution:
http://www.microscopyu.com/tutorials/java/imageformation/airyna/index.html
http://www.microscopyu.com/tutorials/java/lightandcolor/refraction/index.html
2.
Köhler Illumination:
http://www.microscopyu.com/tutorials/java/kohler/index.html
3.
Phase shift of light by an object
http://www.microscopyu.com/tutorials/java/phasecontrast/phasespecimens/index.html
4.
Phase contrast
http://www.microscopyu.com/tutorials/java/phasecontrast/opticaltrain/index.html
http://www.microscopyu.com/tutorials/java/phasecontrast/microscopealignment/index.html
5.
Objectives with adjustable working distance
http://www.microscopyu.com/tutorials/java/aberrations/correctioncollar/index.html
222
Confocal Laser Scanning Microscopy (CLSM)
Problem in conventional microscopy: light, which comes from outside of the focal plane
(above or below) gets to the detector (or eye) and is registered as blur, which decreases the
quality of the image
Solution: A pinhole (iris) is placed into the light path at a position, where it can block out-offocus light. By that means an optical section is imaged (with variable thickness starting with
approx. 0.8 µm) depending on the diameter of the pinhole.
Usually high quality excitation light is needed for that (e.g. coherent laser light with parallel
light beams). The result is a very sharp image without any blur from out-of-focus light with a
slightly higher resolution than with conventional epifluorescence microscopy.
Photomultiplier
confocal pinhole
Laser
dichroic mirror
Scanner
Objective
z-Motor
223
111
Confocal microscopy removes the blur
from thicker objects
http://zeiss-campus.magnet.fsu.edu/tutorials/opticalsectioning/confocalwidefield/index.html
Optical sectioning and 3Dprojections
z-stack
Acquisition of a „z-stack“ (image slices along the zaxis) allows reconstruction of a 3D-projection, which
can be shown as animation
projection
3D rendering
225
112
Spectral imaging
Resolving spectral information on a pixel-by-pixel basis
•
•
•
•
•
•
„Emission finger printing“: emission scan of a
microscopy sample („lambda stack“ of images) at a
given excitation wavelength (e.g. with Zeiss LSM
META systems or with Leica confocal microscopes…)
Alternative: Excitation scan (at a constant emission
wavelength; e.g. using a monochromator light
source)
Combinations of excitation and emission finger
printing (e.g using filter wheels)
Increases the number of markers to be measured in
parallel
Can be used to discriminate fluorophores with
overlapping spectra
Can be used to discriminate specific fluorescence
from autofluorescence
Leica concept
Zeiss META concept
226
Spectral Imaging Confocal Microscopy
(with Emission Curve Analysis)
Leica Confocal Microscope TCS SP2: Monochromator in front of the detector
AOBS: Acousto-Optical Beam Splitter (instead of dicroic mirror)
META System of Zeiss: 32 PMT-detectors every 10.7 nm
(400 – 720 nm): simultaneous wavelength analysis.
lambda-stack
Spectral curve of a
region of interest
227
113
Zimmermann et al.
(FEBS Letters 2003)
Sample with overlapping
fluorophores
1
2
3
4
5
6
7
8
1
2
Emission curves separated
into 8 channels (left) or
2 channels (right)
Equation matrix for the
channel signals based on
reference intensities in the
channels (GFPn and YFPn)
and the unknown
contributions of the
fluorophores
Unmixed fluorescence
(pseudo-coloured)
Spectral imaging example I: CFP, GFP and YFP
http://zeiss-campus.magnet.fsu.edu/articles/spectralimaging/introduction.html
229
114
Spectral imaging example II: strongly
overlapping dyes
SYTOX Green (nucleus), Alexa Fluor
488 conjugated to phalloidin
(filamentous actin network), and
Oregon Green 514 conjugated to goat
anti-mouse primary antibodies
(targeting mitochondria).
Invitrogen Spectra Viewer
http://www.invitrogen.com/site/us/en/
home/Products-andServices/Applications/CellAnalysis/LabelingChemistry/FluorescenceSpectraViewer.html
230
Separation of specific fluorescence from autofluorescence by spectral imaging
231
115
Example for Emission Fingerprinting on a Zeiss
LSM510 META: Separation of GFP and YFP
Acquisition of a
reference lambda
stack for the first
fluorophore (GFP)
Obtain the spectral emission curve for the first fluorophore and
repeat the procedure for the second fluorophore
Intensity
250
YFP
200
GFP
150
100
50
0
500 510 520 530 540 550 560
Emission wavelength (nm)
116
Unmixing of a mixed sample
(GFP-Actin and YFP-membranes)
Emission stack
Unmixed image
Unmixing of signals in pathology samples
(Shown with the Nuance™-Software from Cambridge Research & Instrumentation)
image with mixed signals for
different markers
autofluorescence
Brightfield display
235
117
„Realtime“ confocal microscopy, Spinning disk
confocal microscopy (with Nipkow-disks)
gentle scanning (less bleaching
> good for sensitive life cells
Detection of the signal with a CCDcamera
http://zeiss-campus.magnet.fsu.edu/tutorials/spinningdisk/yokogawa/index.html
236
Companies for confocal microscopes
• Zeiss: http://www.zeiss.de
• Leica: http://www.leica.com
www.confocal-microscopy.com
•
Nikon: http://www.instrumente.nikon.de/
•
Olympus: http://www.olympus.de/microscopy/
237
118
Multiphoton Laser Scanning-Microscopy
A quantum physical phenomenon is used: at very high light
densities (using pulsed lasers, about 900 nm infrared light)
packages of 2 or more photons occur (just in the focal plane !).
These have the same energy as single photons of higher energy
(shoerter wavelength, e.g. 450 nm). Thus these photon packages
can excite a fluorophore, which emits then at for instance 520 nm
(mitted wavelength is horter than the excitation light wavelength !).
An important advantage is that the 900 nm light has a mucher
deeper penetration into tissue (approx. 1 mm), while conventional
excitation can image just down to 0.25 mm. Another advantage is a
reduced overall bleaching effect, as excitation photon packages
occur just in the focal plane.
238
Multiphoton Laser Scanning-Mikroskopie II
conventional excitation
(1-Photon > cone of
ecitation light)
2-Photon excitation:
only a spot of
excitation
239
119
Special Fluorescence Microscopy Techniques
1. FRAP: Fluorescence Recovery After Photobleaching
2. FLIP: Fluorescence Loss in Photobleaching
3. FRET: Fluorescence Resonance Energy Transfer
4. FLIM: Fluorescence Lifetime Imaging Microscopy
5. FISH: Fluorescence In Situ Hybridization
240
FRAP: Fluorescence Recovery After Photobleaching
FRAP at the membrane
Non linear regression analysis
y = span (1-e-kx) + bottom
An image is taken – then a region of the cell is bleached by high laser intensity, followed by
a time series of images after bleaching. Briefly after bleaching the region is significantly
darker and then the fluorescence intensity increases again (fluorescence reoovery) due to
diffusion of molecules into the bleached area. The kinetics of recovery depends on the
diffusion coefficience; the extent of recovery (the plateau to which the fluorescence
recovers) is a measure of the overall mobility (the fraction of mobile molecules versus
molecules immobilized, e.g. to the cytoskeleton)
241
FRAP in the cytosol:
120
inverse FRAP with novel fluorescent
proteins
Protocol: FRAP analysis on Zeiss LSM510
•
•
•
•
•
•
•
•
Capture an image of the whole cell before bleaching
Define a bleaching / scan region (and maybe in addition another scan region that is not
bleached)
Perform a time series with 1 scan prebleach, about 70 iterations of bleaching with 100%
laser power and then 50-100 scans of the bleach region (and also the non-bleached control
region if you specified one)- a good time resolution can only be obtained if just the small
bleach region (and maybe the control region) is scanned - and not the whole cell;
averaging of 2 or 4 scans reduces the electronic noise and leads to better quantifications.
Capture an image of the whole cell after the FRAP time series (with the same conditions as
the prebleach image – for calculating the total loss of fluorescence.
If you want to save disk space: extract the FRAP region and save just this region instead of
the whole image
It is recommended to use the WCIF version of ImageJ for analysis: You can open the LSMfiles with the built-in feature (which also allows opening the time values of the image
series). Measure the mean fluorescence in a control region or for the whole cell for both the
prebleach and the postbleach images and calculate the loss of overall fluorescence due to
the bleaching in the region of interest (this is necessary for obtaining correct recovery
values for the bleach region).
Import the FRAP-image sequence, define a measurement region and apply the „intensity
versus time plot“ plug-in – this will draw a graph of the FRAP curve; clicking the list button,
shows a list of the numerical values (the first 4 parameters are dimension and position of
the region, the rest are the fluorescence intensity values).
Copy the fluorescence raw data from the list to the corresponding column of an Excel
template
243
121
•
Calculate the difference of mean fluorescence from the background and normalize
the fluorescence values to 100% for the initial fluorescence.
•
Divide the percent values by the correction factor calculated from the total loss of
fluorescence (e.g. if total fluorescence decreased from 1 to 0.9 then divide the
mean fluorescence of the FRAP regions for each time value by 0.9 to compensate
for the loss in total fluorescence). A similar compensation can be obtained by
normalizing the FRAP fluorescence values to the control scan region that was not
bleached. This method also compensates more exactly for the bleaching effect in
the course of scanning of the time series (this scanning-dependent bleaching
effect is opposed to the recovery of fluorescence in the bleach region due to
diffusion of non-bleached molecules in the bleach region). This “dynamic
correction” gives a somewhat better estimation of the curve (and the kinetics of
the recovery) – but leads in principle to results that are very similar to the curve
obtained with the “constant correction factor” (by calculating the total loss in
fluorescence based on the intensities of the images that were captured before and
after the FRAP-time series)
•
For non-linear regression analysis (curve fit of the data to a single exponential
association algorithm): Copy the data to a fitting program (such as GraphPad
Prism) and perform the fitting with a “bottom to span” algorithm:
y = span × (1 − e− kx ) + bottom
244
FLIP: Fluorescence Loss in Photobleaching
… to determine the dynamic shuttling of molecules between different
compartments of the cell
A certain compartment A (e.g. the cytosol)
is repetitively bleached by the laser – and
the fluorescence decrease in a different
compartment B is monitored by time lapse
microscopy. Molecules that shuttle from B
to A are bleached in A > thus the
compartment B gets dimmer when there is
a dynamic distribution of molecules
between A and B.
120
cytosol
100
nucleus
80
60
40
20
0
0
2
4
6
122
FLIP to determine a nuclear export signal and a
nucleolar localization signal
NFκB inducing kinase
truncated NIK without the export sequence:
nuclear FLIP
(bleach in nucleus outside nucleoli)
125
nuclear
nucleolar
100
75
50
25
0
0
100
200
300
400
500
600
700
sec
FCS: Fluorescence Correlation Spectroscopy
… to determine diffusion coefficients and interactions between molecules.
The sample is illuminated by the laser at a very small spot, the movements
of molecules in this spot (in and out) cause fluorescence fluctuations, which
are analyzed by correlation functions
123
FRET: Fluorescence Resonance Energy Transfer
Microscopy
Energy can be transferred between two fluorophores when they
are very close to each other (closer than 10 nm) and when the
emission curve of one (the energy donor) overlaps with the
excitation curve of the other one (the acceptor). This transfer of
energy does not happen via photons (!) but by a dipole-interaction
(a quantum physical phenomenon discovered by Theodor Förster
in 1946). As a result the donor fluorophore fluorescence becomes
weaker and the acceptor fluorescence increases.
The FRET effect decreases with the 6th power of the distance; the
distance of half maximal energy transfer is called Förster-Distance
R0 (for CFP and YFP it is approximately 5 nm). As the effect is
usually not detectable anymore at a distance higher than 10 nm it
is ideally suited for monitoring macromolecular interactions
(protein-protein or protein-DNA). By that means, not only the
interaction by itself can be detected, but also the location of the
interaction and its dynamics.
248
Principle of Fluorescence Resonance Energy Transfer
no FRET
Donor
FRET
Acceptor
donor fluor.
(CFP)
acceptor fluor.
(YFP)
E = R06/(R06 + r6) und
nm
excitation
excitation
emission
Donor
Acceptor
R0 = [κ2 × J(λ) × n-4 × Q]1/6 × 970
R0 … Förster-Distance
r ….. real distance
κ ….. Orientation factor
J(λ) … spectral overlap
n … refractive index
Q … Quantum yield of fluor.
249
124
Appropriate Fluorophore Pairs for FRET
Fluorophore Pair
Comments
CFP / YFP
Good combination for normal FRET microscopy using Hglamps as light source and special filters. CFP is poorly
excitated by Ar-lasers, but: good excitation by blue laser
diodes
BFP / GFP
BFP has inferior fluorescence properties
GFP / DsRed-variants
Original DsRed is just fluorescent as tetramer, shows
complex maturation characteristics with green fluorescent
intermediates and tends to aggregate; DsRed2 is a dimer.
The new monomeric DsRed works very fine with GFP.
GFP / YFP
Are very difficult to separate with filters (but can be used in
FLIM and with spectral analysis)
GFP / Cy3 or Alexa 546
Antibodies can be directly labeled with Cy3 or equivalent
Alexa dye and give FRET with a GFP chimera to which they
bind.
FITC / TRITC
Classical FRET pair for labeled proteins (e.g. antibodies)
Alexa 488 / Alexa 546
(Cy3 / Cy5)
alternatives as labeling dyes (superior to FITC and TRITC)
250
FRET can be used to monitor protein-DNA
interactions
spectra of donor and acceptor (GFP-NF-κB
and Tet-labeled DNA, respectively)
1.2
GFP-NFkB (Em)
Tet (Em)
1.0
relative fluorescence
Spektral analysis of a mixture: Increase in
acceptor fluorescence indicates FRET
Tet (Ex)
0.8
0.6
0.4
0.2
Spectral overlap
0.0
480
500
520
540
560
580
600
nm
Excitation of
GFP (488 nm)
GFP-Emission (512 nm)
GFP
GFP-NF-κB
DNA
Tet-Emission (540 nm)
FRET
Tet-label
251
125
FRET can be applied to visualize the interaction
of signaling molecules in living cells
decrease
in Donor
Emission
ECFP and EYFP-Scans
1,2
relative fluorescence
EYFP (Em)
1,0
increase in
Acceptor
Emission
EYFP (Ex)
ECFP (Em)
0,8
ECFP (Ex)
0,6
FRET
0,4
0,2
0,0
350
Spectral overlap
1 nm
400
450
500
550
600
nm
Förster Distanz R0 for ECFP and EYFP: ca. 5 nm (50%
FRET Effizienz): > no FRET-Signal beyond 10 nm.
X
Y
Donor Acceptor
252
Overview of FRET-Microscopy Techniques
1. Acquisition with FRET filter set (donor excitation and acceptor
emission): Problem: coexcitation of the acceptor at the donor
wavelength > false positives
2. Acquisition of a ratio image of acceptor fluorescence at donor
excitation and donor fluorescence at donor excitation
2-Filter FRET Microscopy: Just works when there is equal expression
of donor and acceptor (e.g. in fusion protein, biosensors)
3. 3-Filter FRET Microscopy
4. Determine the kinetics of donor fluorescence bleaching (this is
slower in the presence of a FRET acceptor, as part of the bleaching
energy is transfered to the acceptor)
5. Donor recovery after acceptor photobleaching
6. fluorescence lifetime microscopy– FLIM; fluorescence lifetime of the
donor decreases in the presence of a FRET acceptor
253
126
Spectral crosstalk of donor and acceptor
ECFP and EYFP-Scans
1.2
relative fluorescence
1.0
raw FRET-channel:
0.8
Donor Excitation +
Acceptor Emission
0.6
0.4
0.2
0.0
340 360 380
400
420
440 460
480
nm
Excitation window
of donor
500 520
540
560
580 600
Emission window
of acceptor
Problems:
1.
Co-excitation of the acceptor at the Donor-excitation wavelength >
Non-FRET-Fluorescence in the raw-FRET channel
2.
Signal-overlap of donor into the acceptor channel > Non-FRET
fluorescence in the raw-FRET channel
254
2 Filter-FRET Microscopy (Ratio Imaging)
Ratio of donor emission and acceptor emission at the excitation
wavelength of the donor
Limitations:
•concentration dependent
•donor and acceptor have to
colocalize completely
ECFP and EYFP-Scans
1.2
EYFP (Em)
EYFP (Ex)
relative fluorescence
1.0
ECFP (Em)
ECFP (Ex)
0.8
0.6
0.4
0.2
0.0
350
400
450
500
550
600
nm
just useful for FRETbiosensors with covalent
linkage between donor and
acceptor
(equal-molar expression and
100% colocalization)
excitation
emission-1 emission-2
image = Emission2 : Emission1
255
127
3-Filter FRET Microscopy
3 Images are taken (under constant camera settings):
1. Donor (e.g. CFP-excitation and emission),
2. Acceptor (e.g.YFP-excitation and emission – this signal is not affected by FRET
3. FRET-Filter (raw FRET: CFP-excitation and YFP-emission).
A normalized FRET signal (image) can be calculated by using correction factors
obtained with single stained samples:
FRETc = IFRET - corrCFP x ICFP – corrYFP x IYFP
corrCFP : ca. 0.59
corrYFP : ca. 0.18
CFP / YFP neg. control
CFP-YFP pos. control
256
FRET microscopy example
corrected FRET = IFRET - corrCFP x ICFP – corrYFP x IYFP
sample
Donor
channel
Acceptor
channel
FRET
channel
corr.
factor
corrected
FRET
CFP alone
100
0
60
0.6
0
YFP alone
0
100
20
0.2
0
non-bound
CFP + YFP
100
100
80
0
bound CFP-YFP
100
100
160
80
Donor
neg.
control
Acceptor
corrected FRET normalized FRET
Normalized FRET
(normalized to diff.
expression levels):
=
×
sample
257
128
FRET Microscopy by analyzing the kinetics of
donor bleaching
… this is slowed down in presence of a FRET acceptor
time series of images
time series of images
436 nm
ECFP
FRET
476 nm
EYFP
436 nm
476 nm
ECFP
CFP-Protein alone
CFP- and YFP-Protein
258
Donor-bleaching
kinetics
Probe mit FRET
advantages:
concentration independent
donor and acceptor don‘t have to
colocalize completely
Limitation: requires external control,
difficult to obtain a FRET-image
single exponential decay
y = A 0 .e −kt + offset
Probe ohne FRET
y... Fluor. Signal
A0... starting signal
k... decay constant
t... time
offset... final value
Fluorescence half time Tau: τ = 0.69/k
FRETeff. E = 1 - (τ without
/ τ with Akzeptor.)
259
129
FRET Microscopy by acceptor bleaching and
monitoring donor recovery
(do not use for CFP / YFP)
Donor recovery after acceptor bleaching:
An image of the donor in the presence of
the acceptor is taken, then the acceptor
is bleached (partially), followed by
acquisition of a second donor image
Donor
Donor
FRET
Acceptor
Acceptor
260
Visualisation of biochemical reactions by FRET
microscopy (e.g. phosphorylations)
Detection of the auto-phosphorylation of EGF-receptor on Tyrosine residues using
GFP-EGFR fusion protein and Cy3-labeled anti-P-Tyr antibodies
(donor recovery after acceptor photobleaching technique)
erb2 -P
GFP
Cy3
GFP Ratio image
261
130
FLIM: Fluorescence Lifetime Imaging Microscopy
The lifetime of donor fluorescence (usually in the nanosec. range) is
reduced in presence of a FRET acceptor.
This lifetime can be determined by a special variant of microscopy. Usually a
pulsed or a modulated laser is used for excitation. The fluorescence decay
(Time Domain) or the phase shift (Frequency Domain) of the emission
compared to the excitation is a measure of the fluorescence lifetime.
fluor. image
FLIM image
262
FRET-Biosensors I
Caspase 3-Biosensor:
Apoptosis (Activation of caspase 3) is detected with a CFP-YFP fusion
protein in which CFP and YFP are separated by a caspase 3 cleavage site.
Without apoptosis: FRET, with apoptosis: no FRET
CFP
Caspase 3
-…DEVD…-
YFP
FRET
no FRET
263
131
Other examples for FRET-Biosensors
Calcium-Biosensor: Ca2+-sensitive Calmodulin and a Ca2+/Calmodulin-binding
M13 domain are spliced between CFP and YFP (additional localization
sequences can be added – e.g. signal peptide and ER retention sequence). A
change in the calcium concentration leads to a change in the conformation of
the linker and thus to an alteration of the FRET signal.
Ca2+
YFP
YFP
CFP
FRET
CFP
Calmodulin M13
Ca2+
PKA Activity sensor: CFP and YFP separated by a PKA-substrate sequence
and a 14-3-3 domain, which binds phospho-serine of the PKA substrate
domain.
FRET
PKA-substrate 14-3-3
CFP
YFP
PKA
CFP
YFP
P
264
Digital Image Analyses
Often used: ImageJ (scientific Freeware: http://rsbweb.nih.gov/ij/ )
Different operations can be performed: contrast enhancement,
smoothing, background subtraction, measurement of fluorescence
intensities…
– and further more even math with images can be done (e.g.
dividing one image by another one; each pixel value is divided by
the corresponding pixel value of the second picture – at the same
pixel coordinates)
265
132
Division of images
with ImageJ
266
PixFRET Plugin for ImageJ
133
FRET analysis with self-written ImageJ macro
neg. control
14
12
10
8
6
4
2
0
Negative
Control
IKK1+Myc
IKK2+ Myc
sample
1 sample
2
sample
In Situ Hybridisation (ISH)
… for specific detection of DNA or mRNA sequences.
A labeled DNA- or RNA is hybridized to the target sequence in situ
(in the cell or the tissue) and detected
Applications:
1.
Detection or (semi-) quantification of mRNA
2.
Detection of DNA-sequences in chromosomes (e.g. translocations,
mutations, loss of genes…)
http://www.cytochemistry.net/In_situ.htm
http://osiris.rutgers.edu/~smm/in_situ_hybridization.htm
269
134
Principle of ISH
- a labeled“ probe (e.g. DNA, labeled with Biotin-dUTP by Nick-Translation or an
oligonucleotide, labeled by terminal deoxynucleotidyl transferase, TdT) diffuses
into the cell and hybridizes with the target sequence. Addition of formamide to the
hybridization buffer lowers the specific hybridization temperature, so that at 37°C
only specific target sequences are bound. The probe is then detected via
fluorescence (fluorescence in situ hybr., FISH) or by enzyme activity (e.g. HRP
and colour reaction).
FITC
270
ISH: Advantages and Disadvantages of various probes
Probe type
Advantages
Disadvantages
DNA (double strand)
Easy to use
Subcloning unnecessary
Choice of labeling methods
High specific activity
Possibility of signal amplification
(networking)
Reannealing during hybridization
(decreased probe availability)
Probe denaturation required,
increasing probe length and
decreasing tissue penetration
Hybrids less stable than RNA probes
DNA (single strand)
No probe denaturation needed
No reannealing during hybridization
(single strand)
Technically complex
Subcloning required
Hybrids less stable than RNA probes
RNA
Stable hybrids (RNA-RNA)
High specific activity
No probe denaturation needed
No reannealing
Unhybridized probe enzymatically
destroyed, sparing hybrid
Subcloning needed
Less tissue penetration
Oligonucleotide
No cloning or molecular biology
expertise required
Stable
Good tissue penetration (small size)
Constructed according to recipe
from amino acid data
No self-hybridization
Limited labeling methods
Lower specific activity, so less
sensitive
Dependent on published sequences
Less stable hybrids
Access to DNA synthesizer needed
from: Feldman, RS, Meyer, JS, and Quenzer, LF (1997). Principles of Neuropsychopharmacology. Sunderland, MA: Sinauer Associates, Inc. Pages 31-35.
more recently: BAC probes (can be obtained from collections): high sensitivity for single copy genes… 271
135
Examples for ISH
FISH with interphase nuclei
Fluorescence In Situ-Hybridization on
Metaphase-chromosomes
Detection of a target-mRNA in cryoor paraffin sections
Detection with 33P
Detection with alkal. Phosphatase
272
mRNA-FISH Protocol
•
•
•
•
•
•
•
•
•
•
•
•
Cells are fixed with freshly made 4% formaldehyde in PBS, pH 7.4 for 15 min at
room temperature. All solutions should be made in Molecular Biology grade
ultrapure water (no RNase). Wear gloves at all times and use sterile disposable
pipets and tips.
After rinsing in PBS (3 X 10 min. each), cells are permeabilized with 0.5% Triton
X-100 in 1X PBS for 10 min at 4oC.
Cells are then rinsed in PBS (3 X 10 min. each) and then in 2X SSC (1 X 5 min.).
100 ng of nick translated probe (containing digoxigenin dUTP) and 20 ug of
competitor E. coli tRNA per coverslip are dried down in a Speed Vac (Savant).
This is a good starting place but you may have to titrate your specific probe.
10 μl of deionized formamide is added to the dried DNA.
The probe and tRNA are denatured by heating for 10 min at 90oC. The probe is
chilled on ice immediately.
10 μl of Hybridization buffer (20% dextran sulfate + 4X SSC) is added to the
denatured probe so that the final concentrations in the hybridization mixture
are 5 ng/ml of probe, 1 ug/ml of E. coli tRNA, 2X SSC, and 10% dextran sulfate.
20 μl of hybridization mixture/probe is placed onto each coverslip.
Coverslips are inverted onto a slide and sealed with rubber cement and
incubated in a humid chamber for 16 hrs. at 37oC.
After rinsing in 2X SSC/50% formamide at 37oC, 2X SSC and 1X SSC at room
temperature for 30 min. each, the coverslip containing cells are incubated in 4X
SSC/0.25% BSA/2ug/ml anti-digoxigenin antibody for 60 min. in a humid
chamber at room temperature in the dark.
Coverslips are then rinsed in 4X SSC (1 X 15 min.) at room temperature, 4X
SSC/0.1% Triton X-100 (1 X 15 min.), and 4X SSC (3 X 10 min. each).
Coverslips are mounted in fluorescence mounting medium.
Modified from: Jiménez-García, L. and D.L. Spector. 1993. Cell 73, 47-59.
273
136
Superresolution Microscopy I
STED: Stimulated Emission Depletion
A second laser (depletion laser) „trims“ the excitation spot (point-spread function, PSF) to a
smaller size. Resolution appr. 80 nm.
http://zeiss-campus.magnet.fsu.edu/articles/superresolution/introduction.html
274
Superresolution Microscopy II
Structured Illumination Microscopy (SIM)
A second laser (depletion laser) „trims“ the excitation spot (point-spread function, PSF) to a
smaller size. Resolution appr. 80 - 100 nm.
normal image
SIM image
A known pattern is projected into the
image plane at different angles and
interferes with sample structures,
creating Moiré pattern. Superresolution
information can now be captured by the
microscope from these structures by
mathematical algorithms.
275
(from www.zeiss.de)
137
Superresolution Microscopy
- by single molecule detection
STORM: Stochastic Optical Reconstruction Microscopy using single fluorescent molecules
PALM: Photoactivated Localization Microscopy
Resolution: appr. 30 nm, based on statistical calculation of the center of a Gaussian Fit of a
single molecule. Requires a sensitive camera (e.g. EMCCD: Electron-multiplying chargecoupled device cameras) – and some software, but no specific hardware
http://zeiss-campus.magnet.fsu.edu/articles/superresolution/introduction.html
276
Research Methods - Overview
•
•
•
•
•
•
•
•
cell culture systems
labelling and transfection of cells
analyses of cellular components
analyses of molecular interactions
fluorescence measurements
microscopy
flow analysis (FACS)
analyses of cellular processes (proliferation, apoptosis..)
277
138
Flow Cytometry
Some contents are inspired by “Fluorescence Spectroscopy in
Biological Research”
by Robert F. Murphy
Definitions:
• Flow Cytometry
– Measuring properties of cells in flow
• Flow Sorting
– Sorting (separating) cells based on properties measured in
flow
– Also called Fluorescence-Activated Cell Sorting (FACS)
278
Basics of Flow Cytometry
•Cells in suspension
Fluidics
•flow in single-file
through
•an illuminated volume
where they
Optics
•scatter light and emit
fluorescence
•that is collected,
filtered and
Electronics
•converted to digital
values
•that are stored on a
computer
- Flow Cytometry, Flow Analysis).
- Flow Sorting, Fluorescence
Activated Cell Sorting, FACS
279
139
http://probes.invitrogen.com/resources/education/
280
Fluidics
• Need to have cells in suspension flow in single
file through an illuminated volume
• In most instruments, accomplished by
injecting sample into a sheath fluid as it
passes through a small (50-300 µm) orifice
281
140
Fluidics II
• When conditions are right, sample fluid flows in a central core
that does not mix with the sheath fluid
• This is termed Laminar flow
• Whether flow will be laminar can be determined from
the Reynolds number
V is the mean fluid velocity in (SI units: m/s)
D is the diameter (m)
μ is the dynamic viscosity of the fluid (Pa·s or
N·s/m²)
When Re < 2300, flow is always
laminar
ν is the kinematic viscosity (ν = μ / ρ) (m²/s)
When Re > 2300, flow can be
turbulent
Q is the volumetric flow rate (m³/s)
ρ is the density of the fluid (kg/m³)
A is the pipe cross-sectional area (m²)
282
Flow Cell
Injector
Tip
Fluorescence
signals
Sheath
fluid
Hydrodynamic
Focusing
Focused laser
beam
283
Purdue University Cytometry Laboratories
141
Hydrodynamic Focusing
The figure shows the
mapping between the
flow lines outside and
inside of a narrow
tube as fluid
undergoes laminar
flow (from left to
right). The fluid
passing through cross
section A outside the
tube is focused to
cross section a inside.
284
V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3
Hydrodynamic Focusing II
Example: Focusing of ink by
laminar flow into a capillary
Notice how the ink is focused
into a tight stream as it is drawn
into the tube under laminar
flow conditions.
In flow cytometry:
Sample container and the sheath fluid container are put under defined air
pressure. Laminar flow is maintained (and hydrodynamic focussing is
achieved) by a precise control of the pressure difference between the sample
container, the sheath fluid container and the atmosphere
285
V. Kachel, H. Fellner-Feldegg & E. Menke - MLM Chapt. 3
142
Flow Chamber
Different types are possible,
a commonly used type:
Flow
through
cuvette
(sense in
quartz)
286
H.B. Steen - MLM Chapt. 2
Optics: General scheme
PMT
4
Flow cell
PMT
Dichroic
Filters
3
PMT
2
Bandpass Filters
PMT
1
FSC
Laser
SSC
287
original from Purdue University Cytometry Laboratories;
modified by R.F. Murphy and J. Schmid
143
Forward Scatter (FSC)
When a laser light source is
used, the amount of light
scattered in the forward
direction (along the same axis
that the laser light is traveling)
is detected in the forward
scatter channel
The intensity of forward
scatter is mainly proportional
to the size and surface
properties of cells (or other
particles)
288
Forward Scatter
Detector
Usually not a sensitive PMT detector
(because the light intensity is high)
– but rather a photodiode detector,
which can be set in log10increments (E-1, E0, E1...), and a
linearity gain factor (1.0 – 9.99).
A blocking bar prevents the direct
laser light from hitting the detector.
A predefined FSC-Wert is often used
as threshold to discriminate
between cells and dust particles
289
144
Forward scatter threshold to ignore debris and
small particles or cells
290
• When a laser light source is
used, the amount of light
scattered to the side
(perpendicular to the axis that
the laser light is traveling) is
detected in the side or 90o
scatter channel
• The intensity of side scatter is
mainly dependent on
subcellular structures (e.g.
granules, vesicles…)
Side Scatter
(SSC)
291
145
90 Degree Light Scatter (SSC)
Laser
FSC Detector
90° scattered light
(side scatter) is
detected with a
photomultiplier
detector, where the
sensitivity can be set
in Volt. Amplification
scale can be linear or
logarithmic.
SSC detector
292
Purdue University Cytometry Laboratories
Optics: Fluorescence Channels
dichroic mirrors
bandpass filters
146
Common
Laser
Lines
350
300 nm
457 488 514
400 nm
500 nm
610 632
600 nm
700 nm
Fluorophores
PE-TR Conj.
Texas Red
PI
Ethidium
PE
FITC
cis-Parinaric acid
294
Purdue University Cytometry Laboratories
Sorting of cells
FL1
488 nm Laser
FSC
Sensor
Fluorescence
-
FSC
charged
electrodes
-
+
+
-
Gating
control unit
Sorted
cells
295
Purdue University Cytometry Laboratories; modified
147
296
Sorting Mode
If there are 2 or more cells in one droplet,
and the droplet contains target and non
target cells, then there is a „sorting conflict“,
which has to be solved by defining an
appropriate sorting mode:
Target-Cell
Non-target cell
•
Exclusion-Mode: droplets containing non target cells are dismissed
even if they contain target cells > high purity, maybe lower yield
•
Recovery-Mode: droplets containing target cells are collected, even if
they also contain non-target cells > high yield, maybe low purity.
•
Single Cell Mode: only droplets with single target cells are collected
> high purity, maybe low yield, high counting accuracy.
297
148
Overlap of fluorescence signals and
„Compensation“
298
„Compensation“
When cells are labeled with 2 fluorophores (e.g. FITC and
PE), there might be a signal crosstalk between the detection
channels (dependent on detector voltage settings). This can
be compensated in the follwing way: measuring a sample
containing only fluorophore 1: if there is crosstalk, you see
an elevated intensity in both channels – using the
compensation control of the software, you can bring the
fluorescence signal in the wrong channel down to the
background fluorescence. With a sample stained only with
fluorophore 2 you do the same for the other channel. This
compensation is usually just valid for the detector settings
at which it was set.
compensation for fluorophore 2
299
compensation for fluorophore 1
149
Compensation II
before
compensation
after
compensation
> true double positives can
be determined
300
FACS-Graphs
counts
Histograms: the intensity of one
channel is divided into classes
(often 1024, x-axis) and the
frequency of „events“ (cells)
scored into these 1024 classes.
0
FL1
Dot Plots: 2 different parameters
are plotted on the x- and y-axis;
each cell is a spot on this graph
according to its parameter
intensities.
FL2
Correlation between
histograms and
dotplots
301
150
FACS-Graphs II
FL1
Density Plots: The frequency of events
(cells) in various areas of a dot plot is
colour coded to highlight the peaks
Contour Plots: The frequency of events
(cells) in various areas of a 2D-plot is
visualized by lines representing equal
probability (similar to contour lines of
mountains on a map)
FL2
FL1
FL2
3D-Plots: The frequency of events
measured for 2 parameters is illustrated
by a third axis (z-axis) in a 3D manner.
FL2
302
Statistics and „Gating“
Region R1: 69.3%
Regions: can be defined in
different forms (polygons,
ellipses..). These can be used for
statistics of cells falling into a
certain region – but also for
„gating“ that means rejecting
certain cells inside (or outside) a
certain region for analysis (or
data acquisition) – or for instance
for sorting of cells.
(Polygon-Region)
Marker M1: 67.5%
Marker: Upper and lower limits in
histograms for quantification
Quadrants: split the 2D graph in 4
regions, by the coordinates of the
point where 2 axis intersect.
Allows fast and simple statistics.
UL (upper left): 0.06%
UR (upper right): 29. 88%
LL (lower left): 0.64%
LR (lower right): 69.44%
303
151
Research Methods - Overview
•
•
•
•
•
•
•
•
cell culture systems
labelling and transfection of cells
analyses of cellular components
analyses of molecular interactions
fluorescence measurements
microscopy
flow analysis (FACS)
analyses of cellular processes (proliferation, apoptosis..)
304
Analytical Applications of Flow Analysis
•
•
•
•
•
•
•
•
•
Leukocyte analyses
Phenotyping of cells (CD-Marker)
Immunofluorescence stainings
cell cycle analyses (DNA-content)
Chromosome analyses
Proliferation assays (Brd-U incorporation)
Apopotosis assays (Annexin V, TdT, JC1)
Calcium flux-measurements
etc.
305
152
Leukocyte analyses
Phenotyping of cells
CD8-positive T-cells
CD4-positive T-cells
306
cell cycle analyses by staining of DNA with PI
diploid
Chromosome content
G2M
G0/G1
s
doubled diploid
G0
G1
G0G1
apoptotic
cells
G2/M
S
s
0
Propidium-Iodide Fluorescence of
permeabilized cells after digestion of RNA:
fluor. depends on DNA content
200
400
G2 M
600
4N
2N
DNA Gehalt
800
1000
307
153
Protocol of a Propidium-Iodide Staining
Adherent cells:
trypsinized, suspended in medium + 10% FCS, centrifuged (1000 rpm, 5 min), Pellet
suspended in PBS (1 ml)
Suspension cells:
Centrifuged (1000 rpm, 5 min), Pellet suspended in PBS (1 ml)
Fixation with EtOH:
Pipet cell suspension into 2.5 ml absolute EtOH (final concentration approx. 70%) - or vortex
the suspension at half speed while adding the EtOH) – to prevent clustering of cells during the
fixation. Incubate on ice for 15 min (or over night at –20°C).
Alternative fixation with paraformaldehyde:
Pipet the 1 ml cell suspension into 3 ml 4% paraformaldehyde and fix for 15 min at r.t.
Staining:
Pellet the cells at 1500 rpm for 5 min, Suspend the pellet in 500 µl PI-solution in PBS:
50 µg/ml PI from 50x stock solution (2.5 mg/ml), 0.1 mg/ml RNase A, 0.05% Tritin X-100
Incubate for 40 min at 37°C
Add 3 ml of PBS, pellet the cells (1500 rpm, 5 min) and take off the supernatant
Suspend the pellet in 500 µl PBS for flow analysis
(you can also leave about 500 µl of the diluted staining solution on the pellet and suspend the
cells in this solution > less loss of cells when you take off the sup.) – the rest of the staining
solution does not interfere with the flow analysis.
Flow analysis:
Approximate settings (on FACSort):
FL1: 570 V log. (e.g. if you want to detect GFP)
FL2: 470 V linear
308
Chromosome Analyses
309
154
Analysis of Proliferation
(BrdU-labeling of S-Phase Cells)
Cells are cultured for a given
time in medium containing
Bromodeoxy-Uridin (BrdU). This
nucleotide analogon incororates
into newly synthesized DNA (of
cells in S-phase) – and can be
detected with anti-BrdU
antibodies (e.g. FITC labeled).
FITC-antiBrdU Fluor.
Propidiumjodid Fluor. (PI)
310
Staining of apoptotic cells with JC-1
JC-1 (5, 5´, 6, 6´-tetrachloro-1, 1´, 3, 3´-tetraethylbenzimidazol-carbocyanine iodide)
is a dye, which incorporates in to mitochondrial membranes, where the fluorescence
depends on the membrane potential. In normal cells (with intact mitochondrial
membrane potential) it builds aggregates and emits mainly red fluorescence, in
apoptotic cells (where the membrane potential breaks down) it occurs in monomers
and emits mainly green fluorescence. This can be detected by flow analysis or
microscopy.
311
155
Protocol of a JC-1 Staining of Apoptotic Cells
JC-1 is prepared as a 1000x stock solution in DMSO (5 mg/ml).
For the staining of adherent cells it is diluted in medium to 5 µg/ml (with
vortexing during the dilution to prevent the formation of precipitates); the JC1 containing medium is added to the cells, followed by incubation for 10 min
at 37°C (or RT for 15 min).
Subsequently the cells are washed twice with PBS, trypsinized, suspended in
500 µl PBS and analyzed by flow analysis.
Suspension cells (lymphocytes): suspend 1:1 with 10 µg/ml JC-1 in medium
(final conc.: 5 µg/ml)
Approximate detection settings on FACSort:
FL1: 360 V (log)
FL2: 310 V (log)
Compensation : FL1-7% FL2 und FL2-74% FL1
312
Detection of apoptotic cells by PI-staining of
permeabilized cells (cell cycle analysis)
Fragmented DNA emits
lower fluorescence then
cells with the normal
diploid DNA content – this
„Sub-G0/G1“ population
reflects apoptotic cells
with fragmented DNA )at
a late stage of apoptosis)
313
156
Detection of apoptotic cells with Annexin V
Fluorescence-labeled
Annexin V: binds to
phosphatidylserine, which
is normally on the inner
leaflet of the membrane,
but which is flipped to the
outside during apoptosis
Discrimination between necrotic and
apoptotic cells
PI-pos/Annexin-neg.: necrotic
PI-neg/Annexin-pos.: early apoptotic
PI-pos/Annexin-pos.: late apoptotic, or
necrotic with large holes in the membrane
(where Annexin V can get through)
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Calcium Flux Determination by Flow
Analysis
Cells are stained with a calcium sensitive fluorophore (INDO-1 or
better Fluo4); after a stimulus, the kinetics of the fluorescence signal
is measured (monitoring calcium influx).
315
157
Sources
• Purdue Univ. Cytometry Laboratories
http://www.cyto.purdue.edu/
• Research Institute of Scripps Clinic: http://facs.scripps.edu/
• Invitrogen Tutorials
(http://www.invitrogen.com/site/us/en/home/support/Tutorials.html)
•
•
•
•
BD Tutorials: http://www.bdbiosciences.com/support/training/itf_launch.jsp
J. Paul Robinson (Purdue Univ.)
Robert F. Murphy (Carnegie Mellon Univ., Pittsburgh)
Flow Cytometry and Sorting, 2nd ed. (M.R. Melamed, T.
Lindmo, M.L. Mendelsohn, eds.), Wiley-Liss, New York, 1990 –
abgekürzt: MLM
• Flow Cytometry: Instrumentation and Data Analysis
(M.A. Van Dilla, P.N. Dean, O.D. Laerum, M.R. Melamed, eds.),
Academic Press, London, 1985 – abgekürzt: VDLM
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Companies offering flow analysis equipment
•
•
•
•
•
Becton-Dickinson
http://www.bd.com
Beckman-Coulter
http://www.beckmancoulter.com
Millipore:
http://www.millipore.com/
Accuri (> now part of BD)
http://www.accuricytometers.com/
Partec: http://www.partec.com/
317
158
Free Software: Flowing Software 2
http://www.flowingsoftware.com
318
Flowing Software 2
Quadrants
Regions in histograms
319
159
320
Cell cycle analysis with Flowing Software
•
Define 3 histogram regions (H1, H2, H3:
G0/G1, S and G2/M-phase, respectively)
•
Activate the region control tool: Cell Cycle
•
Define the G2 peak multiplier and peak width
(right click in the cell cycle window)
•
Choose active control (after right clicking)
•
Create statistics (by right clicking into the
histogram window)
•
Create a Stat.List by right-clicking into the
Statistics window
•
Ctrl-N loads the next file, adjusts the H1-H3
regions automatically and calculates the cell
cycle phases (H1-H3)
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160
Tissue Cytometry: Quantitative Image Analysis of
microscopy samples
Single-cell recognition (e.g. based on DAPI-nuclear fluorescence) > generation of cell masks for
quantification of signals (e.g. in different fluorescence channels) > scattergrams can be derived similar to
flow cytometry (for the cells in their original tissue environment!) > Gating and statistics are possible
http://www.tissuegnostics.com
Similar evaluations can be done with
ImageJ using automatic threshold and the
„Analyze particles“ feature.
Tissue Arrays for quantitative comparison of samples
> equal staining conditions for all samples
CA
Normal
PIN
Pat. #1
Pat. #2
Pat. #3
Pat. #4
Control
Gingiva
Cystectomy
Example of a
prostate tissue
array
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Multi-parallel coordinate plots for visualization
of several parameters in parallel
Can be done with Freeware (Mondrian:
http://www.theusrus.de/Mondrian/index.html)
One event (e.g. one cell) is represented by a line linking
several y-axis (for the different parameters e.g.
fluorescence signals); a „population“ can be selected
and is highlighted also for the other parameters. The
data density can be reduced (using a so called alphafactor) to obtain better visibility of numerous data
points.
1
2
3
4
5
6
7
8
9
10
The density of lines (events) can be visualized in colourcoded manner (example: 10-parameter parallel
coordinate plot generated with MatLab) > fast intuitive
visualization of multiparameter data sets
162