1. What is Flow Cytometry? Introduction to Flow Cytometry
IGC Workshop
Applications in Flow Cytometry
Rui Gardner
IGC – April 29, 2010

2. Outline Potential Applications of Flow Cytometry Cell State
Cell Function
Immunophenotyping
Cell activation
Cell cycle
Cell proliferation
Apoptosis
Differentiation
Identification of “stem cells”
Cytokine Secretion
Activation of signalling pathways
Calcium flux
Levels of intracellular reactive oxygen species
Telomere length
Vantagens da Citometria de fluxo
Estatistica populacional
cell sorting
Analise de populaçoes raras
Aplicaçoes
Cell Separation
Sorting

3. Cell Phenotyping

4. Immunophenotyping CD3+ CD4+ CD25- CD3+ CD3+ CD4+ CD3+ CD4+ CD25+ CD3+

5. Cell Activation

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

7. Activation
Activation markers: CD69, CD71, etc

8. Cell Cycle G2 M G1 S G0

9. Cell Cycle DNA content analysis - Propidium Iodide (PI) M G0 G1 G2 S
G2/M
S-phase
Fluorescence (DNA content)

10. Cell Cycle Analysis Cell Cycle Analysis Software Cell Number
G0/G1
Cell Number
G2/M S
Fluorescence Intensity

11. Propidium Iodide plus BrdU staining
Cell Cycle - Bromodeoxyuridine (BrdU) method
Propidium Iodide plus BrdU staining
• Thymidine analog
• Taken up by cells in S-phase
• Usually in combination with Propidium iodide
104
S Phase
103
Anti-BrdU FITC
102
101 G1
G2/M
Propidium Iodide
New Click-It DNA technology from Invitrogen does not require DNA denaturation.

12. Pyronin Y plus Hoechst 33342/33258
Cell Cycle - G0/G1 discrimination
Pyronin Y plus Hoechst 33342/33258
G0/G1 S
G2/M
Cell Count G0 S
G2/M G1
RNA Content
Live Go cell cycle analysis with Hoechst and Pyronin Y
Flow cytometry may be used to determine the level of senescence in a cell population. Go and G1 can be separated by use of Pyronin Y (Sigma) which preferentially binds to RNA and Hoechst (Sigma) which binds to A-T base pairs. Cells are loaded with Hoechst at 10 ug/ml and Pyronin Y at 0.5 ug/ml by incubating for 45 mins at 37C. Pyronin Y is excited at 488 nm and emits at 575 nm, while Hoechst is excited UV lasers ( nm), near UV line (375 nm) or violet diode (405 nm).

13. Apoptosis

14. Cell Death
CELL DEATH – FSC x SSC

15. Propidium Iodide (fixed cells)
Apoptosis
Propidium Iodide (fixed cells)
DNA Degradation

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

17. Annexin V plus propidium Iodide
Apoptosis
Annexin V plus propidium Iodide

18. Analyse by Flow Cytometry
Apoptosis (intracellular staining)
Fix and permeabilize
Add Antibody
Analyse by Flow Cytometry

19. Apoptosis – Bcl-2 family members

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

21. Cell Proliferation

22. Tracking Cell Proliferation with CFSE
Dilution of CFSE
Cell Divisions
STAIN WITH CFSE
CELL
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. 

23. Tracking Cell Proliferation with CFSE
IL-7
IL-7+ DMSO
IL-7+ PI3K Inhibitor
IL-7+ Erk Inhibitor

24. Activation of
Signaling Pathways

25. Phospho-protein detection
Activation of signalling pathways
Phospho-protein detection

26. Activation of signalling pathways

27. Discrimination of High vs. Low responders
Activation of signalling pathways
Discrimination of High vs. Low responders
pStat1
pStat1

28. Activation of signalling pathways
Discrimination of simultaneous vs. non-simultaneous
activation of different pathways in single cells

29. Combining Surface Markers with Phospho-staining

30. Cytokine Secretion

31. Each type of bead coated with capture antibody for particular cytokine
Multiplex Bead Arrays
bead coated with capture antibody for particular cytokine
Cytokines
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
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 Cytokine

32. Multiplex Bead Arrays
NEAT
1/8
1/64
NEG

33. Calcium Flux

34. Calcium Flux
Fluorescence-imaging of human erythrocytes treated with PGE2 using the calcium fluorophor Fluo-4
Effects of T cell receptor stimulation on CD4 cell ionized calcium concentration ([Ca2+]i).

35. Telomere Length

36. Telomere Length

37. Today’s
Future Applications

38. Amnis Image Stream

39. Amnis Image Stream

40. What is Flow Cytometry? Introduction to Flow Cytometry
IGC Workshop
Applications in Flow Cytometry (end)
Rui Gardner
IGC – April 29, 2010

41. Sorting Applications

42. Sorting Immunophenotipic populations
CD3+
CD4+
CD25-
CD25+
CD3+ CD4+ CD25-
CD3+
CD3+ CD4+
CD3+ CD4+ CD25+
Transcriptomics (RNA)
Genomics (DNA)
Metabolomics (metabolites)
Fluorescence microscopy
FISH
Functional Studies
Etc.

43. Establishing Fluorescent Cell Lines
Carina Santos (IMM)
Interphase
Cultured
mCherry signal
Sorted
Anaphase
Human hepatoma cell line
Expressing α-tubulin fused with mCherry
mCherry signal
mCherry signal

44. Chromosome sorting Human cell line with translocation between
chromosome 2 and chromosome 17
Normal human cell line
AT-rich DNA signal
GC-rich DNA signal

45. Establishment of Cell Clones
Sort single cell into each well
time
Clone A
Clone B
Clone C

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