Workshop: Flow Cytometry LBFF: Leeds Bioimaging and Flow Cytometry Facility Workshop – Flow Cytometry: Basic concepts, applications and experimental design
Dec 24, 2015
Workshop: Flow Cytometry
LBFF: Leeds Bioimaging and Flow Cytometry Facility
Workshop – Flow Cytometry:
Basic concepts, applications and experimental design
LBFF: Flow Cytometry Facility Details
Workshop: Flow Cytometry
Location: Garstang level 8
Manager: Dr Gareth Howellhttp://www.fbs.leeds.ac.uk/facilities/flowcytometry/
T: x37270My Office
Workshop: Flow Cytometry
BD FACSCalibur2-laser, 4 colour analyser cytometerFixed emission filter set-up
BD FACSAria2-laser, 7 colour analyser and cells sorting cytometerInterchangable emission filter set-up
Partec PASIIISingle laser, 4 colour analyser cytometerHBO (mercury) lampInterchangable filter set-up
Purpose of this workshop:
To introduce the concepts of flow cytometry (FACS)analysisTo illustrate the role FACS can play in your research
Demonstrate the capabilities of FACSExperimental design
To discuss the limitations of FACS
Seminar:Introduction to FACSApplications available
Practical demonstration:FACS applications and cell sorting
Workshop: Flow Cytometry
• 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
Fluidics
Electronics
Optics(detectors)
Optics(lasers)
Workshop: Flow Cytometry
• 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
FLUIDICS
Workshop: Flow Cytometry
Components of a flow cytometer Electronics
Workshop: Flow Cytometry
LaserLaser
LaserLaser
LaserLaser
Time
Volt
age
Time
Volt
age
Time
Volt
age
Intensity
Cou
nt
h
Low signal height
High signal height
Workshop: Flow Cytometry
Forward scatter
Side scatter
Size and complexity using flow cytometry
Workshop: Flow Cytometry
Cytometer Optical system comprises:
Fluidics
Dichroics and Filters
Detectors
Workshop: Flow Cytometry
FITC FITC
FITC
FITC
FITC
FITC
FITC
FITC
FITC
Emitted fluorescence intensity is proportional to binding sites
FITC
Log scale of Fluorescent Intensity
Num
ber
of E
vent
sFluorescence
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
Workshop: Flow Cytometry
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
Compensation FITC-Fluorescence Overlap
FITC
PE
FITCP
erC
P
650nm 700nm500nm 600nm
Re
lati
ve
In
ten
sit
y
Wavelength (nm)
550nm
PerCP670/LP
FITC 530/30
PE585/42
Workshop: Flow Cytometry
FITC
PE
650nm 700nm500nm 600nm
Re
lati
ve
In
ten
sit
y
Wavelength (nm)
550nm
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.
FITC
PEPerform
Compensation
PerCP670/LP
FITC 530/30
PE585/42
Workshop: Flow Cytometry
Compensation PE-Fluorescence Overlap
FITC
PE
650nm 700nm500nm 600nm
Re
lati
ve
In
ten
sit
y
Wavelength (nm)
550nm 750nm 800nm
PEP
erC
P
PerCP670/LP
FITC 530/30
PE585/42
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 analysisSimple gating stratagies…
Workshop: Flow Cytometry
Whole blood light scatterGate on lymphocytes
(light scatter)Assess T-cell population
(fluorescence)
…to more complex!
Applications of flow cytometry in research
• Immunophenotyping• Stem cell characterisation• Cell cycle• Apoptosis and Cell Viability• Cell proliferation (CFSE, BrdU/Hoechst)• Cell Sorting
Workshop: Flow Cytometry
• Immunophenotypinge.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
Workshop: Flow Cytometry
Stem Cell Characterisation
• Stem Cell Characterisation Functional analysis
• 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
•Cell Cycle Analysis
DNA probes
DAPI }Hoechst } 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
The cell cycle
Note the cell volume (size) and DNA concentration change as the cell progresses
through the cell cycle
Workshop: Flow Cytometry
l
A typical DNA histogram
Stoichiometric DNA probe binding
Workshop: Flow Cytometry
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
Time
Intensity H
H x W = AreaW
Workshop: Flow Cytometry
Cell Cycle Analysis:Bromodeoxyuridine (BrdU) incorporation
•A limitation to standard single colour DNA staining is that we can’t determine whether S-phase cells are actually cycling
•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:
PI
Brd
U-F
ITC S-phase
G1 G2
Limitations. Invitrogen ‘Click-it’ EdU system
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.
Workshop: Flow Cytometry
Apoptosis and Cell Viability
•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
Sub-G1 peakDNA fragmentation allows apoptosis to be quickly assessed with eg. PICan be seen as a population of small peaks to the left of G1 in a histogramQuick and easy way to determine if apoptosis is occurring
Workshop: Flow Cytometry
Annexin-V/PI assay for apoptosis:PS normally on inside of cellular membrane AnnV can bind to externalised PS highlighting cells that are apoptotic PI will only go into cells with compromised membranes – dead (necrotic) cells
AnnV-FITC
PS
XXX
XX
X
PI
Workshop: Flow Cytometry
•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
•Apoptosis – Organelle Analysis
Workshop: Flow Cytometry
(CCCP) carbonyl cyanide m-chlorophenyl hydrazone
•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
Workshop: Flow Cytometry
Alternative: DiOC6(3) for green fluorescent labelled mitochondria
Live/Dead assay
Utilise the properties of dyes that are impermeable to intact cell membranes:
Propidium iodideDAPITOPRO-3
+ve fluorescence indicates compromised cell membranes and therefore dead cells
Yeast cells + TOPRO-3
Dead cells show more granularity and reduced size
Live cells retain their morphology and appear larger in size and less granular
Workshop: Flow Cytometry
• 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
Assessing cell proliferation using flow cytometry
CFSE loaded cells
Workshop: Flow Cytometry
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 labellingDiermeier et al (2004) Cell Prolif. 37:195
• 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%
Workshop: Flow Cytometry
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
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
•Artefacts
•Mis-targetting
•Over expression
•Cell toxicity
•pH sensitive
•Always ensure adequate controls
•N and C terminus constructs
•Check functionality vs WT (if possible)
•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).
UV IRVisible light spectrum
Workshop: Flow Cytometry
Fluorescence - basics
Wavelength (nm)
Fluore
scence
inte
nsi
ty
Fluorescent molecules are characterised by their ability to absorb short wavelength light and emit at a longer wavelength.
Excitation Emission
Workshop: Flow Cytometry
Bandpass eg 530/30 FITC, Alexa 488
Bandpass eg 585/40 Phycoerythrin (PE)
Longpass eg LP670 Cy5, APC, PerCP
UV IRVisible light spectrum
Workshop: Flow Cytometry
Emission SpectraAPC PerCP
Wavelength (nm)400 500 600 700
100%
0%
Nor
mal
ized
Int
ensi
ty
FITC PE
800
Workshop: Flow Cytometry
More Emission Spectra!
APC PerCPPI
Wavelength (nm)400 500 600 700
100%
0%
Cascade Blue
Nor
mal
ized
Int
ensi
ty
FITC PEAlexa 430 PerCP-Cy5.5
800
PE-Cy7
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
Graphic representations of fluorescence data
Green
Red
Green +ve/Red -ve
Green -ve/Red -ve
Green +ve/Red +ve
Green -ve/Red +ve
Workshop: Flow Cytometry