Module C

Isolation and characterization of cell types from whole blood
PBMC cell type distribution using multi-color analysis on FACS-Calibur or FACS-Aria
Cell sorting on FACS-Aria
Analysis of cytokine expression by flow, or ELISA
Techniques for monocyte/macrophage/T cell culture
Model systems of T cell development
Approaches for analysis of impact of disease state, treatment, genotype

Protocol for isolating various cell types from peripheral blood

Mechanism of 4 color detection with FACS Caliber

Compensation of spectral overlap of fluorescent emissions

The FL2 photomultiplier tube (pmt), which detects wavelengths centered around 585 with a bandwidth of 42nm (585/42 orange area) will detect about 30% of the fluorescence of FITC detected by FL1 (530/30, green area): PE = FL2 – 30% FL1

FL1 will detect about 1% of the PE fluorescence detected by FL2: FITC = FL1-1% FL2

FL3 will detect about 3% of the PE fluorescence detected by FL2: TC = FL3-5% FL2

FL2 will detect about 8% of the TC fluorescence detected by FL3: PE=FL2-8% FL3

Fluorescence specifically from PE detected by FL2 = FL2 – 30% FL1 – 8% FL3

These values may change due to efficiency/voltage of pmt’s, laser power, etc.

4-color flow cytometry: Cytokines analyzed within CD3-CD161+cells (NK cells)

4-color flow cytometry analysis of cytokine production. Fresh PBL were cultured 5-h with vehicle (non-stimulated, center panel) or PMA+calcimycin (stimulated, right panel) with monensin added to inhibit cytokine secretion. Cells were then stained with mAb to CD3 (ECD-labeled) and CD56 and CD161 (both CyChrome-labeled); formalin-fixed and permeabilized with saponin buffer; stained with mAb to IL-13 (PE labeled) and IFN-g (FITC-labeled); and analyzed by flow cytometry. Left panel shows a 2-color density plot of CD3 and CD56+CD161 staining with gated lymphocytes (determined from forward and side scatter plots, not shown). IL-13 and IFN-g expression (center and right panels) was analyzed within the gated CD56/CD161+CD3 population (NK cells, outlined region in left plot). Numbers are percent positive cells in indicated quadrants.

Characterization of defective PKA activation specific to T cells in Lupus subjects

Flow cytometry analysis of VASP phosphorylation. Fresh PBL from healthy control subjects (top) and subjects with lupus (bottom) were stained with CD5 mAb (TriColor-labeled) then stimulated 30min with vehicle or 1uM PGE2. Cells were formalin-fixed and permeabilized with saponin buffer, then stained with mAb to phospho-VASP-Ser157 and Goat-anti-mouse IgG (AlexaFluor488-labeled). Left panels show histograms of CD5 expression. Phospho-VASP-Ser157 expression analyzed within grated CD5+ (blue arrows, middle panels) and CD5- (red arrows, right panels) lymphocyte populations. Vehicle, thick line; PGE2, shaded region.