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Laboratory demonstration - DC and T cell activation

Laboratory demonstration - DC and T cell activation. Applications of flow cytometry in basic immunology Generation and characterization of DC Assays for T cell activation Cell proliferation Cell division Cell activation marker. Applications of Flow Cytometry in Basic Immunology.

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Laboratory demonstration - DC and T cell activation

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  1. Laboratory demonstration - DC and T cell activation • Applications of flow cytometry in basic immunology • Generation and characterization of DC • Assays for T cell activation • Cell proliferation • Cell division • Cell activation marker

  2. Applications of Flow Cytometry in Basic Immunology • Basic parameters • Applications • Cell phenotyping • Measurement of cell activation • Visualization of cell division • Detection of intracellular molecues (e.g. cytokine) • Analysis of specific ligand binding • Analysis of intracellular calcium concentration • Analysis of DNA content • Cell sorting

  3. Flow cytometry - the basic parameters • FSC (forward scatter) - Size • SSC (side scatter) - Granularity • FL (flurochrome) – FL-1,FL-2,FL-3 etc.

  4. (1) FSC – the size Forward (parallel) lights

  5. (2) SSC – the granularity Side (angled) lights

  6. (3) Fluorescent-labeled markers Laser light Band Filters Fluorescent detectors FL-1 (green) mirrors FL-2 (red) FL-3 (ultra red)

  7. Fluorochromes & wave length

  8. Cell types of the immune system

  9. Human blood cells analyzed by their FSC and SSC properties Total blood RBC lysed SSC (granularity) FSC (size)

  10. Cell phenotypes & surface markers NK T B

  11. Cluster of Differentiation (CD) markers

  12. Immuno-staining of cell surface markers DirectIndirect

  13. Human blood leukocytes (back-gating analysis) CD14-FITC SSC CD45-PE FSC

  14. Human PBL phenotyping SSC Anti-CD19-PE Anti-CD4-PE FSC Anti-CD3-FITC Anti-CD3-FITC Isotype-PE Anti-CD56/16-PE Anti-CD8-PE Isotype-FITC Anti-CD3-FITC Anti-CD3-FITC

  15. Markers of T cell differentiation

  16. CD4 and CD8 expression on thymocytes, splenocytes and PBL Thymocytes Splenocytes PBL CD4-Cyt5 CD8-FITC

  17. Regulatory T cells (triple stain) SSC CD45RC SBT CT Regulatory T cell: CD45RClo CD4+ CD25+ CD4 CD25 (courtesy of Dr. Lina Tian)

  18. Analysis of cell division by CFSE labeling Cell incubation with CFSE (serum free, 37 0C, 10mins. & wash) CFSE crosses cell membrane & attaches to cytoplasmic proteins Removal of carboxyl groups of CFSE by intracellular esterase Fluorescein Analyed by FC Excitation wave length: 488nm Emitting wave length: 519nm 1 ½ ¼ . . . CFSE:Carboxyfluorescein diacetate Succinimidyl Ester

  19. Visualizing B cell division CFSE intensity

  20. Laboratory demonstration - DC and T cell activation • Applications of flow cytometry in basic immunology • Generation and characterization of DC • Assays for T cell activation • Cell proliferation • Cell division • Cell activation marker

  21. Mitogen (Con A)-induced T cell proliferation (measured by level of DNA synthesis)

  22. Cell activation and proliferation analyzed by the FSC and SSC properties UnstimulatedCon A-stimulated Dot plots SSC (granularity) Density plots FSC (size)

  23. T cell activation analyzedby CD25 (IL-2Ra) expression UnstimulatedCon A-stimulated Cell number CD25-PE SSC FSC

  24. Unstimulated Con A-stimulated CFSE-labeled unlabeled Visualizing T cell activation Cell number SSC (granularity) FSC (size) CFSE intensity

  25. Visualizing & Phenotyping T cell Division A B A B Cell number CD25-PE CFSE intensity

  26. Mitogen-induced T cell proliferation, division vs IL-2Ra (CD25) expression UnstimulatedCon A-stimulated CD25 CFSE intensity

  27. DC:T cell interaction (Huang YG, unpublished data)

  28. - LPS + LPS Isotype Isotype MHC Class II CD40 14.0 67.2 CD80 CD86

  29. DC maturity & APC activity in Mixed Lymphocyte Reaction (MLR) Mature DC Immature DC

  30. Key points for discussions • Requirements for naïve T cell activation? • Key changes during DC maturation? • Association of DC maturity with APC activity? • Induction and measurement of T cell activation – the basis - cell proliferation assay? - indicators of T cell activation? - T cell mitogen? • MLR - peptides presented to T cells in: - allogenic responses? - semi-allogenic responses? - syngenic responses?

  31. GM-CSF GM-CSF+IL-4 GM-CSF + IL-4GM-CSF + IL-4 (TW) (TW + LPS) Isotype Isotype MHC Class II CD40 7.2 14.0 48.7 67.2 CD80 CD86

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