Applications - Espace d`authentification univ

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Introduction to Flow
cytometry and applications

First part : principles

Second part : main applications
– Focused on LEMAR applications

Third part : available equipments in the lab
– FACScalibur Becton-Dickinson
– GUAVA EasyCyte plus
– Utilisation rules
Formation à la cytométrie en flux – 9 novembre 2011
C. Lambert, P. Soudant, N. Le Goïc, C. Paillard, H. hégaret, L. donaghy, M. Auffret
What is flow cytometry?
 Measurement of cell characteristics in a stream of fluid
 A laser beam is focused on the moving cells
 Scattered light and emitted fluorescence are detected
and converted in an electronic pulse
Basic Scheme
Classical flow cytometer
is combining three systems

1- Fluidics
– introduction of the cells in the analyze
chamber

2 - Optics
– Production and collection of scattered
light and emitted fluorescence

3 - Electronics
– Conversion to digital values and storage
on a computer for data analysis
TM
1-Fluidics-FACSCalibur
Need to have cells in suspension flow in single file
through an illuminated volume
Fluidics: hydrodynamic focusing
80 µm
Capillary Guava : 100 µm
needle Facs :
12 µL/min
60 µL/min
Accomplished by injecting sample into a sheath fluid as
it passes through a small (50-300µm) orifice
(laminar coaxial flow - Bernoulli Effect)
Fluidics: hydrodynamic focusing
Sheath fluid
Laminar Coaxial Flow
The Bernoulli Effect
Direction of flow
Lower pressure
Velocity Gradient
Viscous drag along walls.
Particles move to low pressure area
[email protected]
New technologies :
without fluid
Capillary
Fluidics: Sample requirements for
flow cytometry
A suspension of single cells or other particles
in a suitable buffer, usually PBS (phosphate buffer
saline)
Typical density :
105 - 107 cells / ml (FACS)
104 - 5.105 (Guava)
Acquisition speeds : up to 1000 events / sec
typical 300/800 (FACS)
(max 500 : Guava)
Particles size (range) : from ~1 µm to ~40/50 µm
[email protected]
Fluidics : Sample preparation for
flow cytometry
• Suspensions: straightforward samples
• Non-adherent cell cultures
• Circulating cells (blood cells, haemocytes in haemolymph...
• Water borne micro-organisms (zooplankton, phytoplankton…
• Suspensions of bacteria, yeasts, viruses.
[email protected]
Fluidics : Sample preparation for
flow cytometry
•
Sources requiring more preparation
•
.
Adherent cultures and solid tissues have to be
processed to release cells as a suspension
•
•
•
Enzymatic digestion : trypsin, collagenase, pronase.
Chelating agents - removal of ‘binding’ ions : EDTA, EGTA.
Mechanical : teasing, sieving, aspiration (syringing) and
sonication.
•
Chromosomes can be released from mitotic cells
•
Nuclei can also be recovered from tissues stored in
paraffin blocks
[email protected]
2- Optics

Light source (excitation) is constituted of
– Laser beam (blue laser 488nm)
– lens and prisms allow to focus the laser beam

Optical reception is constituted of
– a mirror system and optical filters which direct
specific wavelengths to the corresponding
detector
2- Optics : Basic Optics of a Flow
Laser(s)
Cytometer
Dichroic mirrors
1
2
3
Cell
Collection
Lenses
Scatter
Low & High
angle
Band Pass
Filters
Photomultiplier
tubes
[email protected]
2- Optics: What kind of information are
provided by a flow cytometer?

Relative size (Forward Scatter- FSC) :
diffracted light on the small angle
Nbr of events
Laser
Facs academy
FSC
FACS Academy
Invitrogen
2- Optics: What kind of information
are provided by a flow cytometer?
Internal or complexity relative ‘ granularity ’
(Side Scatter-SSC) : diffracted light on the right angle

SSC
Laser
Invitrogen
Nbr of events
2- Optics: What kind of information are
provided by a flow cytometer?

Relative intensity of
fluorescence
– FL1
– FL2
– FL3
(530 / 30 nm, BP – 525/30 nm BP )
(585 / 42 nm, BP – 583/ 26 nm BP)
(> 670nm, LP – 680/30 nm BP )
Site BD/ Fluorescence Spectrum Viewer
FACS Academy
2- Optics: What kind of information
are provided by a flow cytometer?

Possibility to use several
fluorochromes with
different emission
wavelengths.

Great diversity of
fluorescent probes are
now available
Relative intensity of fluorescence
(FL1, FL2, FL3)

Fluorescence is the property of a molecule to
absorb light of a particular wavelength and
re-emit light of a longer wavelength.
Relative intensity of fluorescence
(FL1, FL2, FL3)

The intensity of the
fluorescence is
proportional to the
number of antibody
fixation sites.
• Applications:
• Cell cycle and ploïdy analysis using DNA staining
• Cell viability, cell functions
• Enzymatic activities
• Quantification of pigments
• etc..
3- Electronics

Creation of a pulse : conversion of the
optical signals into proportional electronic
signal (pulse)
3- Electronics



Analyze height, width, and area of the pulse
Computer interface for data processing
Listed and displayed as Flow Cytometry Standard
(FCS) Files
Interpreting Multi-parameter Data
Single parameters (univariate)
can be displayed as a
histogram.
Dual parameter (bivariate) data
can be displayed in two
dimensions using dot or
density plots.
Three parameters may be
displayed as a 3-D projection,
these are not always
easy to interpret and can often
be ambiguous.
Introduction to Flow
cytometry
(Invitrogen ~12 min video)

http://probes.invitrogen.com/resources/education/t
utorials/4Intro_Flow/player.html
Complementary notions
Fluorescence compensation
Fret
Fluorescence compensation
Compensation is the process
by which the fluorescence
“spillover” originating from
a fluorochrome other
than the one specified
for a particular PMT detector
is subtracted as a percentage
of the signal from other PMT’s.
FITC
PE
FRET
Fluorescence Resonance Energy Transfer
If two fluorescent molecules with suitable spectral overlap
are in close proximity, then the energy from the excited
state of one molecule (the DONOR) can be transferred to
the second molecule (the ACCEPTOR), absorbed and
then re-emitted.
FRET
Fluorescence Resonance Energy Transfer
Typical donor/acceptor pairs:
–
–
–
–
–
Fluorescein / rhodamine
Fluorescein / eosine
Fluorescein / pyrenebutyrate
Anthranilamide / nitrotyrosine
Coumarin / ethidium bromide
Utilisation:
• detect if two labelled protein or nucleic acids come into contact or
a doubly labelled single molecules is hydrolysed
• detect changed in conformation
• measure concentration by a competitive binding assay
Advantages of FCM
High speed analysis
 Multi-parameter data acquisition


Cell sorting
Introduction to Flow
cytometry
(Invitrogen ~12 min video)

http://probes.invitrogen.com/resources/education/t
utorials/4Intro_Flow/player.html
Part II : Applications of Flow
Cytometry
Cellular parameters measurable by flow cytometry.
Without probes : size, shape, cytoplasmic granularity,
autofluorescence and pigmentation.
With probes : DNA content, DNA composition, DNA
synthesis, chromatin structure, RNA, protein, sulphydryl
groups, antigens (surface, cytoplasmic & nuclear), lectin
binding sites, cytoskeletal components, membrane
structure (potential, permeability & fluidity), enzyme activity,
endocytosis, surface charge,receptors, bound and free
calcium, apoptosis, necrosis, pH, drug kinetics, etc...
[email protected]
Most frequent applications in marine
biology and oceanography



Discrimination and identification of
different cell types in various matrix
Assessment of cell viability, cell cycle,
physiological status, cell biochemical
composition
Cell sorting coupled to cell culture or
other assays (PCR)
Applications in marine biology and
oceanography
•
•
•
•
•
•
•
•
•
Cell counting (hemocytes, phytoplancton, bacteria, viruses…)
Cell morphology (size / complexity)
Autofluorescence (pigments: chlorophyll, phycoerythrin…
Mortality / viability
• Mmembrane Integrity (propidium iodide; SytoxGreen®)
• Enzymatic Activities (DCFH-DA)
DNA content (Sybr-green®, propidium iodide)
Oxidative Activity
Apoptosis
mitochondrial membrane potential
…
Bivalve Hemocytes
Detection – counting
bivalves hemocytes
granulocytes
Distinction of hemocyte
subpopulations based on their
size (FSC) and complexity
(SSC)
Hyalinocytes
small agranulocytes
Not that simple !
It requires DNA
staining with
SYBRgreen
(fluorescein) of
oyster hemocytes
R1 = hemocyte
aggregated
R2 = hemocytes (one
cell)
R3 = sperm
Other = cell debris and
unknow stuff
Gate : R1*R2*R3
Gate : R2
granulocytes
Hyalinocytes
Red = aggregates
Green = one cell
Blue = sperm
small agranulocytes
Hemocyte mortality rate

Prodidium iodide (PI) : PI is incorporated only in
cells which had lost their membrane integrity = dead
cells with a orange/red-fluorescence
Orange/ red Fluorescence (PI)
Hemocyte viability
Double staining (SYBR-green + propidium iodide)
FL1
Dead
+ live
Cellules
mortes
hemocytes
et vivantes
FL3
Dead
Cellules
vivantes Cellules
mortes
Live hemocytes
hemocytes
Intracellular
Oxidative Activity
DCFH-DA (dichlorofluorescein diacetate)
(485 nm, 535 nm)
DCFH-DA is membrane permeant,
within the cells the diacetate group is
enzymatically cleaved off (esterase).
Both DCFH-DA and DCFH is nonfluorescent.
In presence of oxidative activity, they are
oxidized to highly fluorescent
dichlorofluorescein.
Intracellular
Oxidative Activity





Anion superoxide : O2Peroxyde d’hydrogène : H2O2
Peroxynitrite (NO3-)
Oxyde nitrique (NO)
Enzymes :
– peroxydase
– xanthine oxydase
– lipoxygenase

cytochrome c
1
4
H
0
Intracellular
Oxidative Activity
a
+
)
3
0
(
L
R
1
2
increasing IOA
S
1
W
granulocytes
3
hyalinocytes
agranulocytes
1
0
0
1
1
0
F
R
S
2
0
1
1
R
F
1
0
0
1
1
0
1
S
2
0
S
1
C
3
Fluorochrome = DCFH-DA (green fluorescence - FL1)
0
-
1
4
H
0
,
Resistant
Sensible
160
140
120
100
80
60
40
Au
gu
st
Se
pt
em
be
r
Ap
ri l
Ju
ly
gametogenesis
20
0
Ju
ne
Activité oxydative
intracellulaire
(fluorescence verte
DCF – FL1)
200
180
M
ay
Oxidative burst (A.U.)
Genetic studies: Effect of genetic stock on oyster
immune system and physiological status;
Lab experiment
 IOA of hemocyte from sensitive ’S’ oysters is higher than the one
from resistant ‘R’ oysters during gametogenesis.
Phagocytosis

Principle/ Method: hemocytes
–
Use of fluorescent latex beads 2 µm:

–
–
Detectable on the FL1 detector of the flow cytometer.
Contact between beads and cells (some hours)
Estimate the
% of active cells (index)
Phagocytosis
events
FL1
Histogram
Phagocytosis

2/3 hours After addition of fluorescent
beads
Gate R1
R1
Gate R1
Phagocytosis
Cytochalasin B
No cytochalasin
Phagocytosis index:
% of active cells (more than 3 beads)
30
25
1 bille (- Cyt B)
20
y = 1,5088x - 2,7631
R2 = 0,9484
15
10
5
0
0
5
10
15
3 billes et plus
20
25
Result : interaction between toxin from algae
and clam hemocytes.
Phagocytose (% hémocytes actifs)
30
Test phagocytose: hémocytes de palourdes / extrait d'Alexandrium
% cellules actives incubation 2h00/ 18°C - 16 mars 2007
(moyenne n=11 ou 12 + int conf p = 0.05)
les lettres indiquent une différence significative, Anova p < 0,0001
b
b
25
20
15
10
a
5
0
contrôle
+ extrait Alexandrium non toxique
Ford, Bricelj, Lambert, Paillard, 2008 (Mar Biol 154, 241-253)
+ extrait Alexandrium toxique
Lysosomes
(Lysotracker red)
Fluorescent probe: LysoTracker® red
• Freely membrane permeant probe that accumulates in
intracytoplasmic lysosomal compartments
Lysosomes:
- intracytoplasmic acidic organelles which contain hydrolases
- can fuse with phagocytic vacuoles (phagosomes)
- resulting intra-vacuolar acidification and contact of lysosomal
enzymes with ingested material favors its degradation.
Example: Turbo cornutus hemocytes lysosomal amount
High
Medium
Low
Donaghy et al., In preparation
Distinction between at least 3
hemocyte populations displaying
low, medium and high amounts
of intracytoplasmic lysosomes.
Differential involvement of
hemocyte subtypes in mollusc
immune response ?
Mitochondrial membrane
potential



The electron chain transport, responsible of the ATP
production, generate a strong electrochemical gradient
across the membrane (negative charge inside).
This
gradient is an indicator of the mitochondrion
functionality and of its energetic status.
To estimate this gradient : use of JC-10 probe (~JC-1)
Mitochondrial membrane
potential (MMP)
JC-10 probe: two iso-forms
- Monomer = green fluo (low MMP)
- Agregates = orange fluo (high MMP)
(1) Cytoplasm loading
Green fluo : monomer
Mitochondrial membrane
potential (MMP): oyster
+ cccp : ratio ~1
Ratio green/ orange > 4
Orange fluo : agregates
CCCP : Carbonyl cyanide 3-chlorophenylhydrazone : decoupling agent
Data from Sébastien Artigaud (Master 2-2010)
DNA content and cell cycle
DNA content of normal cockle
hemocytes
DNA content analysis of neoplastic cells
Normal Haemocytes
Photos: A. Villalba, Spain
4N
<2N
Neoplastic Haemocytes
DNA content analysis of neoplastic cells in
haemolymph of the cockle Cerastoderma edule.
 Examples of heavily diseased cockles
 3N
 6N
 4N
 7N
 5N
 8N
Phytoplancton
Detection – counting
of phytoplancton

Based on the
fluorescence of
pigments (chlorophyll)
Karenia mikimotoi
1) Detection FSC
Complexity (SSC)
Statistics : n = 6
Size (FSC)
Karenia
debris
1) Detection FL3
FL3 (chlorophyll)
Statistics : n = 4
Size (FSC)
Karenia
debris
Nano-pico plancton / cyanobacteria
Chlorophyll autofluorescence
(red)
phycoerythrine autofluorescence
(orange)
Station 4
Charente estuary
Station 3
Station 2
Station 1
Ex 2 : Detection of mixture of 8 eukaryotic cell
types + 2 cyanobacteria
D’après Marie et al., 2004
Ex 3 : coccolithophore counting,
« Size », chlorophyll level.
Gephyrocapsa oceanica
Ex 3 : coccolithophore counting,
« Size », chlorophyll level.
SSC
600
FSC
Volume (µm3)
500
Linéaire (SSC)
y = 6,0728x - 32,317
400
2
R = 0,9337
300
200
100
0
0
10
20
30
40
50
60
70
Echelle relative (FSC ou SSC)
SSC value is a better estimation of cocolithophore volume (R²=0,93) than FSC (R²=0,06)
Viability / mortality
phytoplancton

Principle : membrane integrity loss
– Chlorophyll : red fluorescence, FL3
detector.
– Sytox-green: Yellow-green-fluorescence,
500-530 nm, FL1 detector
SYTOX is incorporated only in cells which had lost
their membrane integrity = dead cells with a Yellowgreen-fluorescence
Viability / mortality
Karenia mikimotoi
Chlorophyll (FL3)
Viability
LIVE CELLS
DEAD CELLS
Karenia mikimotoi
Fluorescence SYTOX
(FL1)
Hansy Haberkorn
Micro-algae: lipid content
Bodipy stainning (493/503)
With copper
Tetraselmis sp.
10µm
without copper
Image: X 100, immersion, epifluorescence HBO,
cube I3 (BP450-490/ BS 500 / LP 515)
green fluorescence (Bodipy)
Autofluorescence of chlorophyll pigments , excited by the laser : red fluo (FL3, 670 LP)
lipid droplet stained with Bodipy : green fluo (FL1, 530/30 BP)
Bacteria, viruses
Bacteria
D’après G. Gregory, BD workshop, Marseille, Avril 2009
EB: ethidium bromide, CFA carboxy-fluorescein-diacetate, Rh rhodamine,
Pi Propidium oidide, CSE : CHEMUNEX Fluorescent Cell Labeling.
Viabilité : Bactérie marine
Vibrio aestuarianus
(pathogène de C. gigas)
Bactéries vivante
31,49%
Sélection taille/ complexité
Membranes endommagées
3,65%
Bactéries "mortes“
64,86%
Double marquage:
Sybr® Green I : ADN/ARN
(fluo verte/jaune, FL1)
Iodure de Propidium:
ADN/ARN
(fluo orange/rouge, FL2) si
perte intégrité membranaire
background
Viruses
D’après G. Gregory, BD workshop, Marseille, Avril 2009
Viruses
(Anne-Claire Baudoux)
12 november 2009
Sybr-green staining
R1 : viruses (in red)
Bacteria
Background
Some other examples of
application in the lab
Oyster Sperms
Grazing experiment
ploidy
Sperm viability : using double staining
(SYBR-green + propidium iodide)
R2 : intact membrane
R3 : damaged membrane
R4 : loss of membrane integrity
Application : interaction
GRAZING
Dunaliella salina
Green micro-algae verte : chlorophyceae
Oxyrrhis sp.
dinoflagellate
GRAZING
Grazing:
Oxyrrhis / Dunaliella
Post doc: Sabine Schultes
Oxyrrhis sp.
Dunaliella salina
Grazing:
Oxyrrhis / Dunaliella
Post doc: Sabine Schultes
suivi concentration en Dunaliella (cell/mL)
120000
concentration (cell/ mL)
103430
99094
107087
108730
100000
80000
84573
Dunaliella + Oxyr.
60000
Dunaliella
40000
45436
20000
1792
652
0
initial
6 heures
24 heures
40 heures
Ploidy (oysters) : diploid
triploid.
On gills cell nucleus : Propidium iodide staining(red fluo FL2)
Ploidy (oysters) : ideally
Standard beads
Ploidy (oysters)
Flow cytometry : main manufacturers
‘FACS’ has become a generic term for ALL flow cytometry
FACS : Fluorescence Activated Cell Sorter
Is actually a trade name of Becton Dickinson (BD).
FACS I, II, III, IV, 400, 420, 440, FACStar, Vantage, DiVa, Aria : Sorters.
FACS Analyser, FACScan, FACSCalibur*, LSR, FACSCanto: ‘Benchtop’.
Coulter (Beckman Coulter) have also produced a
significant range of machines.
EPICS (Electronic Programmable Individual Cell Sorter),
Profile, Elite, XL, ALTRA : a mixture of sorters and ‘benchtops’.
Other Manufacturers : GUAVA, Ortho (Cytofluorograph, Cytoron), Skatron,
Partec, Dako (Galaxy), Cytomation (MoFlo), DakoCytomation : Cyan
benchtop.
LEMAR : two systems
same optics, various fluidics
Facscalibur
Becton Dickinson
GUAVA Easycyte plus®
96 wells micro-plate reader
Fluidic part is reduced
Coming soon (dec. 2011)
Three lasers
10 detectors
Universal loader
…
Utilisation des cytomètres



Personnes « accréditées » = formées
Inscription préalable (calendrier)
Protocole à respecter
– Mise en route
– Sauvegarde des données
– Nettoyage, fermeture
– Bilan d’utilisation mis par écrit
Utilisation des cytomètres

Compensation financière
(coût des appareils : ~80 K€ )
– Entretien hebdomadaire

Nettoyage approfondi, calibrage (billes)
– révision annuelle

Technicien Becton et/ou Guava
– Remplacement des pièces usagées
– Mise à jour des logiciels
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