Flow cytometric analysis of IFN-γ expressed in stimulated human peripheral blood mononuclear cells. HiCK-1 Human Cytokine Positive Control Cells (Cat. No. 555061) were permeabilized with BD Perm/Wash™ Buffer (Cat. No. 554723). The cells were then stained with either Purified Mouse IgG1, κ Isotype Control (Cat No. 550878; dashed line histogram) or with Purified Mouse Anti-Human IFN-γ antibody (Cat No. 550011; solid line histogram) followed by FITC Goat Anti-Mouse IgG/IgM (Cat. No. 555988). Fluorescent histograms showing the expression of IFN-γ (or Ig Isotype Control staining) were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry was performed using a BD LSRFortessa™ system.

Immunocytochemistry: PBMC were isolated from human peripheral blood by density gradient cetrifugation and were cultured for 2 days with plate bound anti-human CD3 and soluble anti-human CD28 in the presence of recombinant human IL-2 (Cat. No. 554603) and recombinant IL-4 (Cat. No. 554605). The cells were subsequently harvested, washed, and recultured with recombinant human IL-2 and recombinant human IL-4 for an additional 3 days. Finally, the cells were harvested, washed, and stimulated with PMA (Sigma, 5 ng/ml) and ionomycin (Sigma, 500 ng/ml) in the presence of GolgiStop™ (Cat. No. 554724) for 4 hours at 37°C. The activated cells were harvested and the level of IFN-γ producing cells were detected by a three-step staining procedure that employs a Purified Mouse Anti-Human IFN-γ antibody(Cat. No. 550011), Biotin Goat anti-mouse IgG secondary antibody (Cat. No. 550337) and Streptavidin-horseradish peroxidase (HRP)( Cat. No. 550946). (Nomarski optics, original magnification 400X).

Flow cytometric analysis of IFN-γ expressed in stimulated human peripheral blood mononuclear cells. HiCK-1 Human Cytokine Positive Control Cells (Cat. No. 555061) were permeabilized with BD Perm/Wash™ Buffer (Cat. No. 554723). The cells were then stained with either Purified Mouse IgG1, κ Isotype Control (Cat No. 550878; dashed line histogram) or with Purified Mouse Anti-Human IFN-γ antibody (Cat No. 550011; solid line histogram) followed by FITC Goat Anti-Mouse IgG/IgM (Cat. No. 555988). Fluorescent histograms showing the expression of IFN-γ (or Ig Isotype Control staining) were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometry was performed using a BD LSRFortessa™ system.