参考图片
Expression of IL-13 by stimulated human CD4+ cells. Isolated human CD4+ cells were stimulated with immobilized anti-human CD3 antibody (UCHT1, 10 µg/ml for plate coating, Cat. No. 555329), soluble anti-human CD28 antibody (20 ng/ml, Cat. No. 555725), 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 washed and subsequently cultured in medium containing IL-2 and IL-4 for 3 days. Finally, the cells were harvested and re-stimulated for 6 hr with PMA (Sigma, Cat. #P-8139; 50 ng/ml), calcium ionophore A23187 (Sigma, Cat. #C-9275; 250 ng/ml) in the presence of GolgiStop™ (2 µM final concentration, Cat. No. 554724). The cells were stained with PE-Cy™5 anti-CD4 (Cat No. 555348) fixed, permeabilized, and subsequently stained with 0.25 µg of PE-rat anti-human IL-13 antibody (Cat No. 562039/554571) by using Pharmingen's staining protocol (see left panel). To demonstrate specificity of staining, the binding of PE rat anti-human IL-13 antibody was blocked by the preincubation of the conjugated antibody with excess recombinant human IL-13 (see middle panel), and by preincubation of the fixed/permeabilized cells with Purified Rat Anti-Human IL-13 antibody (5 µg, Cat No. 554570; see right panel) prior to staining with the PE rat anti-human IL-13 antibody. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control and verified with the recombinant cytokine blocking and unlabeled antibody blocking specificity controls.
Expression of IL-13 by stimulated human CD4+ cells. Isolated human CD4+ cells were stimulated with immobilized anti-human CD3 antibody (UCHT1, 10 µg/ml for plate coating, Cat. No. 555329), soluble anti-human CD28 antibody (20 ng/ml, Cat. No. 555725), 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 washed and subsequently cultured in medium containing IL-2 and IL-4 for 3 days. Finally, the cells were harvested and re-stimulated for 6 hr with PMA (Sigma, Cat. #P-8139; 50 ng/ml), calcium ionophore A23187 (Sigma, Cat. #C-9275; 250 ng/ml) in the presence of GolgiStop™ (2 µM final concentration, Cat. No. 554724). The cells were stained with PE-Cy™5 anti-CD4 (Cat No. 555348) fixed, permeabilized, and subsequently stained with 0.25 µg of PE-rat anti-human IL-13 antibody (Cat No. 562039/554571) by using Pharmingen's staining protocol (see left panel). To demonstrate specificity of staining, the binding of PE rat anti-human IL-13 antibody was blocked by the preincubation of the conjugated antibody with excess recombinant human IL-13 (see middle panel), and by preincubation of the fixed/permeabilized cells with Purified Rat Anti-Human IL-13 antibody (5 µg, Cat No. 554570; see right panel) prior to staining with the PE rat anti-human IL-13 antibody. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control and verified with the recombinant cytokine blocking and unlabeled antibody blocking specificity controls.
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