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

2. LBFF: Flow Cytometry Facility Details Workshop: Flow Cytometry 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

3. 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

4. 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

5. 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

6. 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

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

9. 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

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

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

12. Forward scatter Side scatter Size and complexity using flow cytometry Workshop: Flow Cytometry

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

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

15. 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

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

17. 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

18. Compensation FITC-Fluorescence Overlap FITC PE FITC PerCP 650nm700nm500nm600nm Relative Intensity Wavelength (nm) 550nm PerCP 670/LP FITC 530/30 PE 585/42 Workshop: Flow Cytometry

19. FITC PE 650nm700nm500nm600nm Relative Intensity 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 PE Perform Compensation PerCP 670/LP FITC 530/30 PE 585/42 Workshop: Flow Cytometry

20. Compensation PE-Fluorescence Overlap FITC PE 650nm700nm500nm600nm Relative Intensity Wavelength (nm) 550nm 750nm800nm PE PerCP 670/LP FITC 530/30 PE 585/42 Workshop: Flow Cytometry

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

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

23. …to more complex!

24. 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

25. 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 Workshop: Flow Cytometry

26. Stem Cell Characterisation

27. 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/

28. 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/

29. 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

30. Cell Cycle Analysis

31. 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

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

33. 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 = Area W Workshop: Flow Cytometry

34. 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 BrdU-FITC S-phase G1 G2  Limitations.  Invitrogen ‘Click-it’ EdU system Workshop: Flow Cytometry

35. 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

36. Apoptosis and Cell Viability

37. 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

38. Quick and easy apoptosis assay: Sub-G1 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 Workshop: Flow Cytometry

39. 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 XXXXXX XXXXXX PI Workshop: Flow Cytometry

40. 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 -DiOC 2 (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

41. (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: DiOC 6(3) for green fluorescent labelled mitochondria

42. 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 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

43. 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. 51 Cr-release assay Also cell by cell assay - accurate Single parameter histograms

44. Assessing cell proliferation

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

46. 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

47. 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

48. 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

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

50. 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

51. 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

52. 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

54. Wavelengths of visible light The wavelength of visible light ranges from 380 nm (violet) to 780 nm (red). UVIR Visible light spectrum Workshop: Flow Cytometry

55. Fluorescence - basics Wavelength (nm) Fluorescence intensity Fluorescent molecules are characterised by their ability to absorb short wavelength light and emit at a longer wavelength. Excitation Emission Workshop: Flow Cytometry

56. Bandpasseg 530/30FITC, Alexa 488 Bandpasseg 585/40Phycoerythrin (PE) Longpasseg LP670Cy5, APC, PerCP UVIR Visible light spectrum Workshop: Flow Cytometry

57. Emission Spectra APC PerCP Wavelength (nm) 400500 600 700 100% 0% Normalized Intensity FITC PE 800 Workshop: Flow Cytometry

58. More Emission Spectra! APC PerCP PI Wavelength (nm) 400500 600 700 100% 0% Cascade Blue Normalized Intensity FITC PE Alexa 430 PerCP-Cy5.5 800 PE-Cy7 Workshop: Flow Cytometry

59. 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

60. 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