参考图片
Expression of TNF by stimulated CD4+ and CD4- BALB/c spleen cells. After 6 h stimulation with hamster anti-mouse CD3 (Clone 145-2C11, 2 µg/ml, Cat. No. 553057) and hamster anti-mouse CD28 (Clone 37.51, 2 µg/ml, Cat. No. 553294) antibodies in the presence of BD GolgiStop™ (Cat. No. 554724; aka monensin, 3 µM) the splenocytes were stained with mouse BD FcBlock™ (1 µg/1 million cells; Cat No. 553142), then 0.06 µg of PE-conjugated rat anti-mouse CD4 (PE-RM4-5, Cat. No. 553049). The cells were then fixed, permeabilized, and subsequently stained with 0.06 µg of FITC-conjugated rat anti-mouse TNF antibody (FITC-MP6-XT22, Cat. No. 554418, left panel). To demonstrate specificity of staining, the binding of the FITC-MP6-XT22 antibody was blocked by preincubation of the antibody conjugate with recombinant mouse TNF (0.06 µg, Cat. No. 554589; middle panel), and by preincubation of the fixed/permeabilized cells with unlabeled MP6-XT22 antibody (2 µg, Cat. No. 554416; right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking (middle panel) and antibody blocking (right panel) specificity controls.
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