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 Calcium fluxCalcium flux Levels of intracellular reactive oxygen speciesLevels of intracellular reactive oxygen species Immune reconstitution studiesImmune reconstitution studies Minimal Residual DiseaseMinimal Residual Disease

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

3. Activation CD69, CD71, etc

4. 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.

5. STAIN WITH CFSE DILUTION OF CFSE Cell Divisions

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

7. Cell proliferation using Ki-67 staining

8. Cell Cycle Analysis DNA content analysis - Propidium Iodide

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

10. 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

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

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

13. Apoptosis Bcl-2 family members Bcl-2

14. CELL DEATH – FSC x SSC

15. Apoptosis Propidium Iodide (fixed cells)

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

17. Apoptosis Annexin V plus propidium Iodide

18. Apoptosis Annexin V plus propidium Iodide

19. 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

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

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

22. Activation of signalling pathways

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

24. Combining surface markers with Phospho-staining