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

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Presentation on theme: "What is Flow Cytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry IGC – April 29, 2010."— Presentation transcript:

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

2 Outline Potential Applications of Flow Cytometry 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 Sorting Cell State Cell Function Cell Separation

3 Cell Phenotyping

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

5 Cell Activation

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

7 Activation Activation markers: CD69, CD71, etc

8 Cell Cycle G2 M G1 S G0

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

10 Cell Cycle Analysis Cell Cycle Analysis Software G 0 /G 1 S G 2 /M Fluorescence Intensity Cell Number

11 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 New Click-It DNA technology from Invitrogen does not require DNA denaturation. Anti-BrdU FITC G1 G2/M S Phase Propidium Iodide

12 Cell Cycle - G0/G1 discrimination Pyronin Y plus Hoechst 33342/33258 G0/G1 S G2/M G0 S G2/M G1 Cell Count RNA Content

13 Apoptosis

14 Cell Death CELL DEATH – FSC x SSC

15 Apoptosis Propidium Iodide (fixed cells) DNA Degradation

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

17 Apoptosis Annexin V plus propidium Iodide

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

19 Apoptosis – Bcl-2 family members

20 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

21 Cell Proliferation

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

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 Activation of signalling pathways Phospho-protein detection

26 Activation of signalling pathways

27 Discrimination of High vs. Low responders 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 Multiplex Bead Arrays bead coated with capture antibody for particular cytokine Cytokines Amount Cytokine

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

33 Calcium Flux

34 Effects of T cell receptor stimulation on CD4 cell ionized calcium concentration ([Ca 2+ ] i ). Fluorescence-imaging of human erythrocytes treated with PGE 2 using the calcium fluorophor Fluo-4

35 Telomere Length

36

37 Today’s Future Applications

38 Amnis Image Stream

39

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

41 Sorting Applications

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

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

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


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