Flow Cytometry Workshop

1. Flow Cytometry Workshop Insert Date Dr Gareth Howell g.j.howell@leeds.ac.uk x37270

2. Workshop: Flow Cytometry LBFF: Leeds Bioimaging and Flow Cytometry Facility Workshop – Flow Cytometry: Basic concepts, applications and experimental design

3. 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

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

10. 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

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

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

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

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

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

20. FITC 530/30 PE 585/42 PerCP 670/LP Workshop: Flow Cytometry Perform Compensation PE PE FITC FITC 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 600nm 650nm 700nm 550nm Wavelength (nm)

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

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

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

24. …to more complex!

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

26. 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. Immunophenotyping e.g. diagnosis of leukaemia Workshop: Flow Cytometry 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. Workshop: Flow Cytometry • 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

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

32. H H x W = Area Intensity W Time 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

33. S-phase BrdU-FITC G2 G1 PI 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: hLimitations. hInvitrogen ‘Click-it’ EdU system

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

36. Apoptosis and Cell Viability

37. 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)

38. Workshop: Flow Cytometry Sub G1 apoptosis assay 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. 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. AnnV-FITC PS PI X X X X X X 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

41. 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. 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 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. 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%

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

48. Workshop: Flow Cytometry Multiplex beads

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