Workshop: Flow Cytometry
LBFF: Leeds Bioimaging
and Flow Cytometry Facility
Workshop – Flow Cytometry:
Basic concepts, applications and
experimental design
Workshop: Flow Cytometry
LBFF: Flow Cytometry Facility Details
Location: Garstang level 8
Manager: Dr Gareth Howell
http://www.fbs.leeds.ac.uk/facilities/
flowcytometry/
E: g.j.howell@leeds.ac.uk
T: x37270
My Office
Workshop: Flow Cytometry
BD FACSCalibur
2-laser, 4 colour analyser cytometer
Fixed emission filter set-up
BD FACSAria
2-laser, 7 colour analyser and cells sorting cytometer
Interchangable emission filter set-up
Partec PASIII
Single laser, 4 colour analyser cytometer
HBO (mercury) lamp
Interchangable filter set-up
Workshop: Flow Cytometry
Purpose of this workshop:
To introduce the concepts of flow cytometry (FACS)analysis
To illustrate the role FACS can play in your research
Demonstrate the capabilities of FACS
Experimental design
To discuss the limitations of FACS
Seminar:
Introduction to FACS
Applications available
Practical demonstration:
FACS applications and cell sorting
Workshop: Flow Cytometry
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What is flow cytometry?
Components
Size and complexity using flow cytometry
Fluorescence and Multicolour flow cytometry
Cell cycle analysis
Apoptosis and necrosis assay
Cell proliferation assay
Sorting
Workshop: Flow Cytometry
• What is flow cytometry?
– The analysis of single particles, often cells,
within a heterogeneous suspension
• Whole blood, Cell cultures, Separated tissue,
Isolated nuclei, Bacteria/yeast/parasites, Algae &
plankton
• Signal from individual particles is collected for
analysis as they pass through a laser in a stream
of fluid.
• Data displayed as events on histograms/dot plots
Workshop: Flow Cytometry
Workshop: Flow Cytometry
Components of a flow cytometer
Electronics
Fluidics
Optics
(lasers)
Optics
(detectors)
Workshop: Flow Cytometry
FLUIDICS
• Vital that cells pass through the
laser bean in single suspension
• Cells injected into a flowing
stream of saline solution (sheath
fluid)
• Hydrodynamic focusing
• Compresses cell stream to
approx 1 cell diameter
• Allows single cells to be
interrogated by the laser
•Optimal ‘imaging’ of cells is
achieved with a ‘low’ flow rate
and high concentration of
sample
Workshop: Flow Cytometry
Components of a flow cytometer
Electronics
Laser
Voltage
Workshop: Flow Cytometry
Low signal height
High signal height
Laser
Voltage
Time
Laser
Count
Voltage
Time
h
Time
Intensity
Workshop: Flow Cytometry
Size and complexity using flow cytometry
Side
scatter
Forward
scatter
Workshop: Flow Cytometry
Cytometer Optical system comprises:
Dichroics and Filters
Fluidics
Detectors
Workshop: Flow Cytometry
Fluorescence
Emitted fluorescence intensity is proportional to binding sites
FITC
FITC
FITC
FITC
Number of Events
FITC
FITC
FITC
FITC
Log scale of Fluorescent Intensity
FITC
FITC
Workshop: Flow Cytometry
FACS machines use lasers as sources for excitation; fixed single wavelength. Fluorescent light
emission collected using filters as before. Therefore have to use flurophores compatible with
lasers employed: FACSCalibur/FACSAria 488 and 647nm lasers.
APC
Workshop: Flow Cytometry
Emission is collected through emission filters positioned within the optical system of the flow
cytometer.
APC
Dyes suitable for use on flow cytometers:
• 488 excitation:
– FITC, Alexa 488, GFP, YFP
– PE, PI, RFP,
– PerCP, 7-AAD, PE-Cy5, PE-Cy7
• 633nm excitation:
– APC, TOPRO-3, Cy5, Cy7
Workshop: Flow Cytometry
Compensation FITC-Fluorescence Overlap
PE
585/42
PerCP
670/LP
Relative Intensity
PE
FITC
530/30
PerCP
FITC
500nm
550nm
600nm
650nm
Wavelength (nm)
700nm
FITC
Workshop: Flow Cytometry
Perform
Compensation
FITC
PE
585/42
PerCP
670/LP
Relative Intensity
FITC
530/30
FITC
24.8% of the FITC signal subtracted from PE.
On a FacsCalibur flow cytometer, there is no
provision to subtract FITC signal from PerCP,
referred to as cross-beam compensation.
500nm
550nm
600nm
650nm
Wavelength (nm)
700nm
Workshop: Flow Cytometry
Compensation PE-Fluorescence Overlap
PerCP
670/LP
Relative Intensity
PE
FITC
PE
530/30 585/42
PerCP
FITC
500nm
550nm
600nm
650nm
Wavelength (nm)
700nm
750nm
800nm
PE
Workshop: Flow Cytometry
Optimal
Compensation
Under
Compensation
Over
Compensation
16-colour compensation possible now on latest 3-laser, multi-parameter cytometers
Workshop: Flow Cytometry
Applying Gates for sub-population analysis
Simple gating stratagies…
Whole blood light scatter
Gate on lymphocytes
(light scatter)
Assess T-cell population
(fluorescence)
…to more
complex!
Workshop: Flow Cytometry
Applications of flow cytometry in research
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Immunophenotyping
Stem cell characterisation
Cell cycle
Apoptosis and Cell Viability
Cell proliferation (CFSE, BrdU/Hoechst)
Cell Sorting
Workshop: Flow Cytometry
• Immunophenotyping
e.g. diagnosis of leukaemia
COMBINATION POPULATION IDENTIFIED
CD4+/CDw29+ Helper/effector, more mature memory
cells
CD4+/CD45R+ Suppressor inducer, less mature
non-memory cells
CD4+/Leu8+ Suppressor inducer, some helper
function
CD4+/Class II MHC Activated cells, immature cells
CD4+/CD25+ Activated cells (IL2 receptor)
CD4+CD38+ Immature cells, activated cells
CD8+/CD11b+ Of the CD11b+ cells the suppressors
are bright CD8+ and NK are dim
CD8+ CD8+/CD28+ Cytotoxic precursor/effector cells
CD8+/CD57+ Cytotoxic function
CD8+/Class II MHC+ Activated cells, immature cells
CD8+/CD25+ Activated cells (IL2 receptor)
CD8+/CD38+ Immature cells, activated cells
CD16+/CD57+ Low NK activity
CD16+/CD56+ Most potent NK activity
Stem Cell Characterisation
• Stem Cell Characterisation
Functional analysis
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Cytosolic aldehyde dehydrogenase (ALDH) activity
High levels found in stem cells
Drug resistance
Cleavable enzyme assay (AldeFluor, StemCell Tech.)
http://science.cancerresearchuk.org/
• Stem Cell Characterisation
Side population analysis
• Efficient membrane pumps
• Exclude dyes e.g. Rhodamin 123
and Hoechst dye
• Hoechst dyes bind DNA in live
cells (blue and red fluorescence)
• UV excitation
• Pumped out by ABC (ATPase
Binding Cassette)
• Stem cells can be characterised
by low side populations –ve for
Hoechst dye.
• Membrane markers to confirm.
http://science.cancerresearchuk.org/
• Stem Cell Characterisation
Clinical Application – CD34+ Stem Cell Enumeration
• Method of repopulating stem cells
following radiotherapy treatment
• Patient treated to produce excessive
levels of pluripotent cells which are
harvested from peripheral blood
• Number of cells reintroduced
important in succsss rate of procedure
• Abs vs stem cell markers CD34 and
CD45 used in enumeration procedure
Cell Cycle Analysis
Workshop: Flow Cytometry
•Cell Cycle Analysis
DNA probes
DAPI
Hoechst
The cell cycle
}
}
UV
Propidium iodide (PI) }
7-AAD
}
488
TOPRO-3
DRAQ5
}
}
633
These dyes are stoichiometric –
number of bound molecules are
equivalent to the number of DNA
molecules present
Note the cell volume (size) and DNA
concentration change as the cell progresses
through the cell cycle
Workshop: Flow Cytometry
Stoichiometric DNA probe binding
l
A typical DNA histogram
Workshop: Flow Cytometry
H
H x W = Area
W
Time
Measuring height against width gives us
area
Two G1 cells together will have the same
PI intensity as a G2 cell, but the area
(signal h x w) will be greater and therefore
can be discriminated on a plot of signal
width vs area
Workshop: Flow Cytometry
•A limitation to standard single colour DNA
staining is that we can’t determine whether Sphase cells are actually cycling
BrdU-FITC
Cell Cycle Analysis:
Bromodeoxyuridine (BrdU) incorporation
S-phase
G2
G1
•Cells take up BrdU during S-phase, but not
during G1 or G2, an Ab vs BrdU then allows
us to determine which cells are actively
cycling within a population by two-colour
analysis:
hLimitations. hInvitrogen ‘Click-it’ EdU system
PI
Workshop: Flow Cytometry
Pulse-label with BrdU and
taking samples at specific time
points allows us to determine
how cells behave kinetically
through the cell cycle.
Apoptosis and Cell Viability
Workshop: Flow Cytometry
•Apoptosis
• Gene directed cell death
• An event that occurs during development and a response to trauma or disease
• Cancer cells develop a strategy to evade apoptosis
Apoptosis results in a number of
cellular events that can be analysed
by FACS:
•Fragmentation of DNA (subG1
assay, Hoechst dyes)
•Membrane structure and integrity
Annexin-V, PI)
•Mitochondrial function (Mitotracker
Red)
•Caspase activity (antibodies assay)
Workshop: Flow Cytometry
• Quick and easy apoptosis assay: Sub-G1
DNA fragmentation allows apoptosis to
be quickly assessed with eg. PI
Can be seen as a population of small
peaks to the left of G1 in a histogram
Quick and easy way to determine if
apoptosis is occurring
Sub-G1 peak
Workshop: Flow Cytometry
Annexin-V/PI assay for apoptosis:
hPS normally on inside of cellular membrane hAnnV can bind to externalised PS
highlighting cells that are apoptotic hPI will only go into cells with compromised
membranes – dead (necrotic) cells
AnnV-FITC
PS
PI
Workshop: Flow Cytometry
•Apoptosis – Organelle Analysis
•Membrane potential of the organelle reduced
•Mitochondrial activity appears to change in parallel with cytoplasmic
and plasma membrane events
•Dyes that accumulate in mitochondria can therefore play role in
detecting apoptosis
-Mitotracker Red CMXRos
-JC-1
-DiOC2(3)
-Laser Dye Styryl-751 (LDS-751)
•Reagent combinations can provide a window on intracellular processes
not available with the muchused pairing of annexin V and propidium
iodide
Workshop: Flow Cytometry
•Mitotracker Red can be loaded into live cells and taken up by mitochondria
•Loss of membrane potential causes apoptoic cells to loose dye from organelle
•Shift in fluorescence intensity indicates compromised mitochondria
(CCCP) carbonyl cyanide m-chlorophenyl hydrazone
Alternative: DiOC6(3) for green fluorescent labelled mitochondria
Workshop: Flow Cytometry
Yeast cells + TOPRO-3
Live/Dead assay
Utilise the properties of dyes that are
impermeable to intact cell
membranes:
Propidium iodide
DAPI
TOPRO-3
+ve fluorescence indicates
compromised cell membranes and
therefore dead cells
Dead cells show more
granularity and reduced
size
Live cells retain their
morphology and
appear larger in size
and less granular
• Cell mediated cytotoxicity assay
• Dye exclusion assay to assess
cell death, PKH26 (Sigma)
• Example: tumour cells (target)
and NK cells (effector)
• Positive cytotoxic event
recorded as an increase in cell
fluorescence
• No requirement for
radioisotopes e.g. 51Cr-release
assay
• Also cell by cell assay accurate
Single parameter histograms
Assessing cell proliferation
Workshop: Flow Cytometry
Assessing cell proliferation using flow cytometry
CFSE loaded cells
Assessing cell proliferation using flow cytometry
BrdU/Hoechst quenching assay
DNA binding dye Hoechst
fluorescence quenched if BrdU
incorporated into DNA
Can be used to assess cell
proliferation
PI not quenched – allows
determination of cell cycle as before.
Requires flow cytometer with UV excitation
Needs careful optimization of BrdU labelling
Diermeier et al (2004) Cell Prolif. 37:195
Workshop: Flow Cytometry
• Cell sorting
– Allows rare populations to
be isolated from
heterogenous populations
(cell culture, blood
samples, etc)
– Can isolate sub cellular
particles (e.g. endosomes,
nucleus, chromosomes)
– Allows transfection
experiments to be enriched
and single cell clones to be
isolated
– Can produce purity >95%
Cell Sorting Chromosomes
• Chromosome specific DNA libraries,
DNA for sequencing, probes for reverse
painting, array painting.
• Many lymphomas have chromosomal
abnormalities.
• Base specific dyes allow
chromosomes to be separated on dot
plots
http://www.chrombios.com/Service/ServiceFACS.html
Workshop: Flow Cytometry
Fluorescent proteins and their applications in bioimaging
Workshop: Flow Cytometry
What can we do with fluorescent
proteins?
•Use as reporter genes to identify gene activation
•Study transfection rates / success
•Expression of tagged proteins
-Placed in-frame with gene of interest
•Compare expression / localisation against function (combine FACS with
imaging)
•Environmental indicators (pH)
•Protein-protein interactions (FRET, split-GFP)
Workshop: Flow Cytometry
Disadvantages of fluorescent
proteins?
•Size
•Always ensure adequate controls
•Artefacts
•N and C terminus constructs
•Mis-targetting
•Check functionality vs WT (if possible)
•Over expression
•Cell toxicity
•pH sensitive
•Don’t always select/gate brightest cells! Be
objective
•Stable cell lines? Transgenics?
•Alternative expression vector
Workshop: Flow Cytometry
• Summary
– Flow cytometry is a powerful method for rapidly
quantitating cellular fluorescence
– A number of functional assays such as cell cycle and
apoptosis can be determined by flow and can be used
as a method for assessing e.g. the effects of drugs on
cell function, or the expression of mutant proteins
– Finally, cells and sub-cellular particles can be sorted
from heterogeneous samples to yield near
homogeneous populations for subsequent culturing or
analysis.
Workshop: Flow Cytometry
Wavelengths of visible light
The wavelength of visible light
ranges from 380 nm (violet) to
780 nm (red).
Visible light spectrum
UV
IR
Workshop: Flow Cytometry
Fluorescence - basics
Excitation
Emission
Fluorescence intensity
Fluorescent molecules are
characterised by their ability to absorb
short wavelength light and emit at a
longer wavelength.
Wavelength (nm)
Workshop: Flow Cytometry
Visible light spectrum
UV
IR
Bandpass
eg 530/30
Bandpass
eg 585/40
Longpass
eg LP670
FITC, Alexa 488
Phycoerythrin (PE)
Cy5, APC, PerCP
Workshop: Flow Cytometry
Emission Spectra
100%
PE
APC
PerCP
Normalized Intensity
FITC
0%
400
500
600
Wavelength (nm)
700
800
Workshop: Flow Cytometry
More Emission Spectra!
Cascade Blue FITC Alexa 430
PE
PI APC PerCP PerCP-Cy5.5 PE-Cy7
Normalized Intensity
100%
0%
400
500
600
Wavelength (nm)
700
800
Workshop: Flow Cytometry
Designing Multicolour Experiments
Why?
•Allows a number of different structures (proteins / lipids / compartments) to
be visualized at the same time
• Can provide clues / evidence to the function of your protein of interest
• Design principles can be applied to any fluorescent molecule: fluorescent
protein, membrane marker, antibody or dye in live or fixed cells
How?
• Simply by studying the configuration of the imaging system and the
excitation / emission characteristics of the proposed dyes one can design a
multicolour fluorescent experiment
Workshop: Flow Cytometry
Red
Graphic representations of fluorescence
Green -ve/
Green +ve/
data
Red +ve
Red +ve
Green
Green -ve/
Red -ve
Green +ve/
Red -ve