1. Potential Applications of Flow Cytometry Cell activation statusCell activation status Cell cycle distributionCell cycle distribution Cell divisionCell division ApoptosisApoptosis Differentiation stateDifferentiation state Cytokine SecretionCytokine Secretion Activation of signalling pathwaysActivation of signalling pathways

2. Potential Applications of Flow Cytometry Calcium fluxCalcium flux Levels of intracellular reactive oxygen speciesLevels of intracellular reactive oxygen species Identification of “stem cells”Identification of “stem cells” Telomere lengthTelomere length......

3. Potential Applications of Flow Cytometry Cell activation statusCell activation status Cell cycle distributionCell cycle distribution Cell divisionCell division ApoptosisApoptosis Differentiation stateDifferentiation state Cytokine SecretionCytokine Secretion Activation of signalling pathwaysActivation of signalling pathways

4. Activation IL-7 67% Medium 23% Activation FSC x SSC – Cell size

5. Activation CD69, CD71, etc

6. Potential Applications of Flow Cytometry Cell activation statusCell activation status Cell cycle distributionCell cycle distribution Cell divisionCell division ApoptosisApoptosis Differentiation stateDifferentiation state Cytokine SecretionCytokine Secretion Activation of signalling pathwaysActivation of signalling pathways

7. Cell Cycle Analysis DNA content analysis - Propidium Iodide

8. Cell Cycle Analysis DNA content analysis - Propidium Iodide

9. Cell Cycle Analysis DNA content analysis - Propidium Iodide Careful with doublets! DNA Content G0/G1 S Phase G2/M Or doublets of G0/G1 cells?

10. Cell Cycle Analysis DNA content analysis - Propidium Iodide Careful with doublets!

11. Cell Cycle Analysis DNA content analysis - Propidium Iodide G 0 -G 1 S G 2 -M Fluorescence Intensity Cell Number

12. Cell Cycle Analysis Propidium Iodide plus BrdU staining Thymidine analog Taken up by cells in S-phase Usually in combination with Propidium iodide Cell cycle analysis - Bromodeoxyuridine (BrdU) method New Click-It DNA technology from Invitrogen does not require DNA denaturation.

13. Cell Cycle Analysis Propidium Iodide plus BrdU staining Propidium Iodide Anti-BrdU FITC G1 G2/M S Phase

14. Cell Cycle Analysis Pyronin Y plus Hoechst 33342/33258 – G0-G1 discrimination

15. Potential Applications of Flow Cytometry Cell activation statusCell activation status Cell cycle distributionCell cycle distribution Cell divisionCell division ApoptosisApoptosis Differentiation stateDifferentiation state Cytokine SecretionCytokine Secretion Activation of signalling pathwaysActivation of signalling pathways

16. Tracking cell divisions with CFSE CFSE: Carboxy-fluorescein diacetate, succinimidyl ester (fluorescein molecule containing a succinimidyl ester functional group and two acetate moieties) Diffuses freely into cells Intracellular esterases cleave the acetate groups converting CFSE into a fluorescent, membrane impermeant dye. Not transferred to adjacent cells. Retained by the cell in the cytoplasm Does not adversely affect cellular function During each round of cell division, the relative intensity of the dye is decreased by half.

17. STAIN WITH CFSE DILUTION OF CFSE Cell Divisions

18. IL-7 IL-7+ DMSO IL-7+ PI3K Inhibitor IL-7+ Erk Inhibitor

19. Potential Applications of Flow Cytometry Cell activation statusCell activation status Cell cycle distributionCell cycle distribution Cell DivisionCell Division ApoptosisApoptosis Differentiation stateDifferentiation state Cytokine SecretionCytokine Secretion Activation of signalling pathwaysActivation of signalling pathways

20. CELL DEATH – FSC x SSC

21. Apoptosis Propidium Iodide (fixed cells)

22. Apoptosis Annexin V-fluorochrome plus Propidium Iodide (non-fixed cells)

23. Apoptosis Annexin V plus propidium Iodide

24. Apoptosis Annexin V plus propidium Iodide

25. (intracellular staining) Fix and permeabilize Add Antibody Analyse by Flow Cytometry Apoptosis Bcl-2 family members

26. Apoptosis Bcl-2 family members Bcl-2

27. Apoptosis Bcl-2 family members Flow cytometric analysis of Raji cells, using Bim Antibody (IF Preferred) (blue) compared to a nonspecific negative control antibody (red). Bim But …

28. Apoptosis Activated forms of caspases Flow cytometric analysis of Jurkat cells, untreated (blue) or etoposide-treated (green), using Cleaved Caspase-3 (Asp175) Antibody (Alexa Fluor® 488 Conjugate). Untreated Etoposide

29. Potential Applications of Flow Cytometry Cell activation statusCell activation status Cell cycle distributionCell cycle distribution Cell DivisionCell Division ApoptosisApoptosis Differentiation stateDifferentiation state Cytokine SecretionCytokine Secretion Activation of signalling pathwaysActivation of signalling pathways

30. Analysis of T cell development CD4-CD8-CD3- (TN) CD4+CD8-CD3- (ISP) CD4+CD8+CD3-/lo (DP) CD4+CD8+CD3+ (TP) CD4+ CD8-CD3+ (SP4) CD4- CD8+CD3+ (SP8)

31. Potential Applications of Flow Cytometry Cell activation statusCell activation status Cell cycle distributionCell cycle distribution Cell DivisionCell Division ApoptosisApoptosis Differentiation stateDifferentiation state Cytokine SecretionCytokine Secretion Activation of signalling pathwaysActivation of signalling pathways

32. Cytokine Secretion (intracellular cytokine staining) Activate T cell Add inhibitor of protein export (ex: Brefeldin A; Golgi-STOP) Fix and permeabilize Add Cytokine- Specific Antibody Analyse by Flow Cytometry

33. Cytokine Secretion (intracellular cytokine staining) anti-hIL-2 Ab

34. Stimulate + Brefeldin A Incubate Wash and fix Stain for surface antigens Wash Permeabilize Wash Stain with cytokine- specific antibodies Intracellular Cytokine Staining Plus Surface Staining

35. Intracellular Cytokine Staining Plus Surface Staining IFN-  CD8 / CD69

36. Potential Applications of Flow Cytometry Cell activation statusCell activation status Cell cycle distributionCell cycle distribution Cell DivisionCell Division ApoptosisApoptosis Differentiation stateDifferentiation state Cytokine SecretionCytokine Secretion Activation of signalling pathwaysActivation of signalling pathways

37. Activation of signalling pathways (phospho-protein detection)

38. Activation of signalling pathways

39. Discrimination of High vs. Low responders P-STAT6

40. Activation of signalling pathways

41. Discrimination of simultaneous vs. non-simultaneous activation of different pathways in single cells

42. Combining surface markers with Phospho-staining

44. PFA/saponin protocol failed to detect P-STAT5 (but...better for surface staining and better cell recovery) Activation of signalling pathways IMPORTANCE OF FIXATION METHOD

45. Analyse by Flow Cytometry Telomere length by Flow FISH Tetramer-CY5 (and/or Ab) + Crosslink to cell surface Denature +Probe Blood 2001 vol 97, 700 Hybridise Permeabilise

47. CD45RA Telomere CD4 + CD45RA + gate CD4 + CD45RA - gate Telomere Telomere Length in naïve and memory CD4+ T cells

48. MULTIPLEXED BEAD ARRAYS Can use arrays of beads to measure multiple cytokines at once on the one sample Can use arrays of beads to measure multiple cytokines at once on the one sample Each type of bead coated with capture antibody for particular cytokine Each type of bead coated with capture antibody for particular cytokine Mix beads with sample, wash, then add anti- cytokine ab with fluorchrome reporter Mix beads with sample, wash, then add anti- cytokine ab with fluorchrome reporter Amount of fluorescence measured proportional to amount of cytokine bound to each bead (equivalent to ELISA OD reading) Amount of fluorescence measured proportional to amount of cytokine bound to each bead (equivalent to ELISA OD reading)

49. NEAT1/8 Y-axis FL3 discriminates 5 bead types X-axis is reporter fluorescence for amount of cytokine bound to bead 1/64NEG

50. Y-axis FL3 discriminates 5 bead types X-axis is reporter fluorescence for amount of cytokine bound to bead MOUSE 1 MOUSE 2

51. Future Advances More colours for immunofluorescence (quantum dots, tandem dyes) More colours for immunofluorescence (quantum dots, tandem dyes) Reduced laser size and capillary flow techniques mean smaller instruments Reduced laser size and capillary flow techniques mean smaller instruments Instruments can now image cell at point of laser interrogation Instruments can now image cell at point of laser interrogation

52. Amnis Image Stream