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What is Flow Cytometry ?

What is Flow Cytometry ?. Introduction to Flow Cytometry. IGC Workshop. Applications in Flow Cytometry. Rui Gardner. IGC – April 29, 2010. Outline. Potential Applications of Flow Cytometry. Cell State. Cell Function. Immunophenotyping Cell activation Cell cycle

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What is Flow Cytometry ?

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

  2. Outline Potential ApplicationsofFlowCytometry CellState CellFunction • Immunophenotyping • Cell activation • Cellcycle • Cell proliferation • Apoptosis • Differentiation • Identification of “stem cells” • Cytokine Secretion • Activation of signalling pathways • Calcium flux • Levels of intracellular reactive oxygen species • Telomere length CellSeparation • Sorting

  3. Cell Phenotyping

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

  5. CellActivation

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

  7. Activation Activation markers: CD69, CD71, etc

  8. CellCycle M G0 G1 G2 S

  9. CellCycle DNA content analysis - PropidiumIodide (PI) M G0 G0/G1 G1 G2 G2/M S S-phase Fluorescence (DNA content)

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

  11. CellCycle- Bromodeoxyuridine (BrdU) method Propidium Iodide plus BrdU staining • Thymidineanalog • Taken up by cells in S-phase • Usually in combination with Propidium iodide 104 S Phase 103 102 Anti-BrdU FITC 101 G1 G2/M Propidium Iodide NewClick-It DNA technologyfromInvitrogen does notrequire DNA denaturation.

  12. CellCycle - G0/G1 discrimination Pyronin Y plus Hoechst 33342/33258 G0/G1 G0 G1 S G2/M S CellCount G2/M RNA Content

  13. Apoptosis

  14. CellDeath 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. Add Antibody Analyse by Flow Cytometry Apoptosis (intracellularstaining) Fix and permeabilize

  19. Apoptosis – Bcl-2 familymembers

  20. Apoptosis – ActivatedformsofCaspases Untreated Etoposide FlowcytometricanalysisofJurkatcells, untreated (blue) oretoposide-treated (green), usingCleaved Caspase-3(Asp175) Antibody (AlexaFluor® 488 Conjugate).

  21. CellProliferation

  22. Dilution of CFSE Cell Divisions TrackingCellProliferationwith CFSE STAIN WITH CFSE CELL

  23. IL-7 IL-7+ DMSO IL-7+ ErkInhibitor IL-7+ PI3K Inhibitor TrackingCellProliferationwith CFSE

  24. Activationof SignalingPathways

  25. Activationofsignallingpathways Phospho-protein detection

  26. Activationofsignallingpathways

  27. pStat1 Activationofsignallingpathways Discrimination of High vs. Low responders pStat1

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

  29. CombiningSurfaceMarkerswithPhospho-staining

  30. CytokineSecretion

  31. MultiplexBeadArrays bead coated with capture antibody for particular cytokine Cytokines Amount Cytokine

  32. MultiplexBeadArrays NEAT 1/8 1/64 NEG

  33. CalciumFlux

  34. CalciumFlux 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. WhatisFlowCytometry? Introduction to Flow Cytometry IGC Workshop Applications in Flow Cytometry(end) Rui Gardner IGC – April 29, 2010

  41. SortingApplications

  42. SortingImmunophenotipicpopulations CD3+ CD4+ CD25- CD25+ CD3+ CD4+ CD25- CD3+ CD3+ CD4+ CD3+ CD4+ CD25+ Transcriptomics (RNA) Genomics (DNA) Metabolomics (metabolites) Fluorescencemicroscopy FISH FunctionalStudies Etc.

  43. Interphase Anaphase EstablishingFluorescentCellLines Carina Santos (IMM) Cultured mCherrysignal Sorted Human hepatoma cell line Expressing α-tubulinfusedwithmCherry mCherrysignal mCherrysignal

  44. Chromosomesorting Human cell line with translocation between chromosome 2 and chromosome 17 Normal humancellline AT-rich DNA signal GC-rich DNA signal

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

  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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