1. Flow Cytometry Workshop
Insert Date
Dr Gareth Howell
x37270

2. LBFF: Leeds Bioimaging and Flow Cytometry Facility
Workshop: Flow Cytometry
LBFF: Leeds Bioimaging and Flow Cytometry Facility
Workshop – Flow Cytometry:
Basic concepts, applications and experimental design
Basics: light microscopy, fluorescence and epifluorescence, image resolution,
Confocal microscopy
Digital deconvolution microscopy
Digital images basics
Multicolour experimental design
Fluorescent dyes for labelling cellular structures
Fluorescent proteins in experiments

3. LBFF: Flow Cytometry Facility Details
Workshop: Flow Cytometry
LBFF: Flow Cytometry Facility Details
Location: Garstang level 8
Manager: Dr Gareth Howell
flowcytometry/ E:
T: x37270
My Office

4. Workshop: Flow Cytometry
BD FACSAria
2-laser, 7 colour analyser and cells sorting cytometer
Interchangable emission filter set-up
BD FACSCalibur
2-laser, 4 colour analyser cytometer
Fixed emission filter set-up
Partec PASIII
Single laser, 4 colour analyser cytometer
HBO (mercury) lamp
Interchangable filter set-up

5. Purpose of this workshop:
Workshop: Flow Cytometry
Purpose of this workshop:
To introduce the concepts of flow cytometry (FACS)analysis
To illustrate the role flow can play in your research
Demonstrate the capabilities of flow
Experimental design
To discuss the limitations of flow
Seminar:
Introduction to flow
Applications available
Practical demonstration:
flow applications and cell sorting

6. What is flow cytometry (FACS or FCM)? Components
Workshop: Flow Cytometry
What is flow cytometry (FACS or FCM)?
Components
Light scatter parameters
Fluorescence and Multicolour
Cell cycle analysis
Apoptosis and necrosis assay
Cell proliferation assay
Sorting

7. 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

8. Workshop: Flow Cytometry

9. Workshop: Flow Cytometry
Electronics
Components of a flow cytometer
Fluidics
Optics
(detectors)
(lasers)

10. FLUIDICS 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

11. Workshop: Flow Cytometry
Electronics
Components of a flow cytometer

12. Workshop: Flow Cytometry
Laser
Time
Voltage
Low signal height
High signal height
Count h
Intensity

13. Size and granularity using flow cytometry
Workshop: Flow Cytometry
Size and granularity using flow cytometry
Forward scatter
Side scatter

14. Workshop: Flow Cytometry
Cytometer Optical system comprises:
Dichroics and Filters
Fluidics
Detectors

15. Fluorescence Workshop: Flow Cytometry
Emitted fluorescence intensity is proportional to binding sites
FITC
FITC
FITC
FITC
FITC
FITC
FITC
FITC
FITC
FITC
Number of Events
Log scale of Fluorescent Intensity

16. 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.

17. Workshop: Flow Cytometry
Emission is collected through emission filters positioned within the optical system of the flow cytometer.

18. 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
* tandem dyes

19. Compensation FITC-Fluorescence Overlap
Workshop: Flow Cytometry
Compensation FITC-Fluorescence Overlap
FITC
530/30 PE
585/42
PerCP
670/LP
FITC PE
PerCP
Relative Intensity
500nm
550nm
600nm
650nm
700nm
Wavelength (nm)

20. Workshop: Flow Cytometry
Perform
Compensation PE PE
FITC
FITC
FITC
530/30 PE
585/42
PerCP
670/LP
Relative Intensity
24.8% of the FITC signal subtracted from PE.
On a FacsCalibur flow cytometer, there is no provision to subtract FITC signal from PerCP.
500nm
550nm
600nm
650nm
700nm
Wavelength (nm)

21. Compensation PE-Fluorescence Overlap
Workshop: Flow Cytometry
Compensation PE-Fluorescence Overlap
FITC
530/30 PE
585/42
PerCP
670/LP PE
FITC
Relative Intensity
PerCP
500nm
550nm
600nm
650nm
700nm
750nm
800nm
Wavelength (nm) PE

22. Optimal Compensation Under Compensation Over Compensation
Workshop: Flow Cytometry
Optimal
Compensation
Under
Compensation
Over
Compensation
16-colour compensation possible now on latest 3-laser, multi-parameter cytometers

23. Assess T-cell population
Workshop: Flow Cytometry
Applying Gates for sub-population analysis
Simple gating stratagies…
Gate on lymphocytes
(light scatter)
Assess T-cell population
(fluorescence)
Whole blood light scatter

24. …to more complex!

25. Applications of flow cytometry in research
Workshop: Flow Cytometry
Applications of flow cytometry in research
Multicolour analysis
Cell cycle
Cell proliferation
Apoptosis and Cell Viability
Cell Sorting
Multiplex analysis

26. Applications of flow cytometry in research
Workshop: Flow Cytometry
Applications of flow cytometry in research
Multicolour analysis
Immunophenotyping
Cells surface antigen detection (e.g. receptors, adhesion molecules)
Intracellular staining
Assessing infection/transfection levels
Antibodies/ dyes/ Quantum dots

27. e.g. diagnosis of leukaemia
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

28. 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

29. Cell Cycle Analysis

30. Cell Cycle Analysis Workshop: Flow Cytometry 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

31. Workshop: Flow Cytometry
Stoichiometric DNA probe binding
A typical DNA histogram l

32. Workshop: Flow Cytometry
Time
Intensity H
H x W = Area W
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

33. Bromodeoxyuridine (BrdU) incorporation
Workshop: Flow Cytometry
Cell Cycle Analysis:
Bromodeoxyuridine (BrdU) incorporation PI
BrdU-FITC
S-phase G1 G2
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:
hLimitations. hInvitrogen ‘Click-it’ EdU system

34. Assessing cell proliferation: BrdU incorporation
Workshop: Flow Cytometry
Assessing cell proliferation: BrdU incorporation
Pulse-label with BrdU and taking samples at specific time points allows us to determine how cells behave kinetically through the cell cycle.

35. Assessing cell proliferation using flow cytometry
Workshop: Flow Cytometry
Assessing cell proliferation using flow cytometry
CFSE loaded cells

36. Apoptosis and Cell Viability

37. Apoptosis Workshop: Flow Cytometry 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)

38. Sub G1 apoptosis assay Workshop: Flow Cytometry Sub-G1 peak
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

39. Apoptosis detection using viability dye uptake
Workshop: Flow Cytometry
Apoptosis detection using viability dye uptake
Changes in membrane permeability due to apoptosis allow intracellular dyes to stain unfixed cells
7-AAD (DNA)
Live cells exclude dye
Apoptotic cells stain 7-AADdim
Dead cells stain 7-AADbright

40. 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 X PI

41. Apoptosis – Organelle Analysis
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 much used pairing of annexin V and propidium iodide

42. Workshop: Flow Cytometry
Mitotracker Red can be loaded into live cells and taken up by mitochondria
Loss of membrane potential causes apoptotic 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

43. Live/Dead assay Workshop: Flow Cytometry Yeast cells + TOPRO-3
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
Live cells retain their morphology and appear larger in size and less granular
Dead cells show more granularity and reduced size

44. 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

45. Cell Sorting

46. Cell sorting Workshop: Flow Cytometry
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%

47. Cell Sorting Transfected Cells
Workshop: Flow Cytometry
Cell Sorting Transfected Cells
Improve transefection efficiency
siRNA knock down
Stable cell line production
Rare population isolation
Single cell cloning
Isolate spcific cell types from tissue preps
Up to 4 populations simultaneously
Various collection tubes and plates

48. Workshop: Flow Cytometry
Multiplex beads

49. Fluorescent capture bead technology
Workshop: Flow Cytometry
Fluorescent capture bead technology
Beads of various fluorescent intensities
Can be conjugated with antibodies or biotin
Multiplex conjugated kits
Bender MedSysytems
Beckton Dickinson
Beckman Coulter
Luminex
Qiagen
Upstate
ELISA principals

50. Workshop: Flow Cytometry
Coated latex bead
(FL1)
FL1
FL2
Analyse by flow cytometry using bivariant dot plot
Incubate with e.g. cell lysate Y
Incubate with FL2 labelled antibody vs protein of interest

51. Fluorescent proteins and their applications in bioimaging
Workshop: Flow Cytometry
Fluorescent proteins and their applications in bioimaging

52. What can we do with fluorescent proteins?
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)

53. Disadvantages of fluorescent proteins?
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

54. 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.