参考图片
Expression of IL-3 by stimulated human CD4 cells. Isolated human CD4+ cells were stimulated with immobilized anti-human CD3 mouse antibody (UCHT1, Cat. No. 555329), soluble anti-human CD28 mouse antibody (CD28.2, Cat. No. 555725), recombinant human IL-2 (10 ng/ml final concentration; Cat. No. 554603) and recombinant human IL- 4 (20 ng/ml final concentration; Cat. No. 554605) for 2 days. The cells were subsequently cultured in medium containing recombinant human IL-2 and recombinant human IL- 4 for 3 days. Finally, the cells were harvested and re-stimulated for 6 hours with PMA (Sigma, Cat. #P-8139) and calcium ionophore A23187 (1 µg/ml final concentration; Sigma, Cat. #C-9275) in the presence of Golgi-Stop™ (2 µM final concentration; Cat. #554724). The cells were harvested, stained with Pe-Cy5 anti- CD4 (RPA-T4, Cat. No. 555348), fixed, permeabilized, and subsequently stained with 0.12 µg of PE-rat anti-human IL-3 antibody (PE-BVD3-1F9, Cat. No. 554676) (see Left panel). To demonstrate specificity of staining, the binding by PE- BVD3-1F9 antibody was blocked by each of the following: 1) preincubation of the fluorochrome-conjugated antibody with 0.5µg recombinant human IL-3 (see Center panel) and by 2) preincubation of the fixed/permeabilized cells with unlabeled BVD3-1F9 antibody (5 µg; Cat. No. 554673; see Right panel) prior to staining with the PE-BVD3- 1F9 antibody. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabelled antibody blocking specificity controls.
Expression of IL-3 by stimulated human CD4 cells. Isolated human CD4+ cells were stimulated with immobilized anti-human CD3 mouse antibody (UCHT1, Cat. No. 555329), soluble anti-human CD28 mouse antibody (CD28.2, Cat. No. 555725), recombinant human IL-2 (10 ng/ml final concentration; Cat. No. 554603) and recombinant human IL- 4 (20 ng/ml final concentration; Cat. No. 554605) for 2 days. The cells were subsequently cultured in medium containing recombinant human IL-2 and recombinant human IL- 4 for 3 days. Finally, the cells were harvested and re-stimulated for 6 hours with PMA (Sigma, Cat. #P-8139) and calcium ionophore A23187 (1 µg/ml final concentration; Sigma, Cat. #C-9275) in the presence of Golgi-Stop™ (2 µM final concentration; Cat. #554724). The cells were harvested, stained with Pe-Cy5 anti- CD4 (RPA-T4, Cat. No. 555348), fixed, permeabilized, and subsequently stained with 0.12 µg of PE-rat anti-human IL-3 antibody (PE-BVD3-1F9, Cat. No. 554676) (see Left panel). To demonstrate specificity of staining, the binding by PE- BVD3-1F9 antibody was blocked by each of the following: 1) preincubation of the fluorochrome-conjugated antibody with 0.5µg recombinant human IL-3 (see Center panel) and by 2) preincubation of the fixed/permeabilized cells with unlabeled BVD3-1F9 antibody (5 µg; Cat. No. 554673; see Right panel) prior to staining with the PE-BVD3- 1F9 antibody. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabelled antibody blocking specificity controls.
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