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Expression of IL-4 by stimulated human peripheral blood mononuclear cells (PBMC). Human PBMC were stimulated with soluble anti-human CD3 antibody (1 µg/ml; Cat. No. 555329), recombinant human IL-2 (10 ng/ml, Cat. No. 554603) and recombinant human IL-4 (10 ng/ml; Cat. No. 554605) for 2 days. The cells were subsequently cultured in medium containing IL-2 and IL-4 for 3 days. Finally, the cells were harvested and stimulated for 6 h with PMA (Sigma) and calcium ionophore A23187 (Sigma) in the presence of BD GolgiStop™ (Cat. No, 554724). The cells were harvested, stained with FITC anti-CD4 (Cat. No. 555346) fixed, permeabilized, and subsequently stained with 0.05 µg of APC-rat anti-human IL-4 antibody (APC-MP4-25D2, Cat. No. 554486) by using BD Biosciences Pharmingen's staining protocol (left panel). To demonstrate specificity of staining, the binding of APC-MP4-25D2 antibody was blocked by preincubation of the fixed/permeabilized cells with unlabelled MP4-25D2 antibody (2.5 µg, Cat. No. 554482; right panel) prior to staining. The quadrant markers for the bivariate dot plot were set based on the autofluorescence controls and verified using unlabelled antibody blocking controls. This APC-conjugated reagent can be used in any flow cytometer equipped with a a dye, HeNE or red diode laser. These include the dual laser BD FACStarPLUS™, BD FACSVantage™ or BD FACSCalibur.™
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