Applications of flow cytometry in basic immunology Generation and characterization of DC Assays for T cell activation –Cell proliferation – Cell division.

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

–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 Applications of Flow Cytometry in Basic Immunology

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

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

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

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

Fluorochromes & wave length

Cell types of the immune system

Human blood cells analyzed by their FSC and SSC properties FSC (size) SSC (granularity) RBC lysedTotal blood

Cell phenotypes & surface markers TBTB NK

Cluster of Differentiation (CD) markers

Immuno-staining of cell surface markers DirectIndirect

Human blood leukocytes (back-gating analysis) CD14-FITC CD45-PEFSC SSC

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

Markers of T cell differentiation

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

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

CFSE: Carboxyfluorescein diacetate Succinimidyl Ester Analysis of cell division by CFSE labeling Cell incubation with CFSE (serum free, 37 0 C, 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 1½¼...1½¼...

Visualizing B cell division CFSE intensity

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

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

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

T cell activation analyzed by CD25 (IL-2R  ) expression CD25-PE Cell number Unstimulated Con A-stimulated SSC FSC

Visualizing T cell activation CFSE intensity Cell number SSC (granularity) FSC (size) Unstimulated Con A-stimulated CFSE-labeledunlabeled

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

Mitogen-induced T cell proliferation, division vs IL-2R  (CD25) expression CFSE intensity CD25 Unstimulated Con A-stimulated

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

Isotype MHC Class II CD80 CD86 CD40 - LPS + LPS

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

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?

Isotype MHC Class II CD80 CD86 CD40 GM-CSF GM-CSF+IL-4 GM-CSF + IL-4 GM-CSF + IL-4 (TW) (TW + LPS)