参考图片
Expression of IL-4 by stimulated CD4+ and CD4-BALB/c spleen cells. BALB/c spleen cells were cultured for 72 h in medium containing Staphylococcus aureus enterotoxin B (2 µg/ml; Sigma, St. Louis, MO), recombinant mouse IL-2 (10 U/ml, Cat. No. 550069) and recombinant mouse IL-4 (2 ng/ml, Cat. No. 550067). The cells were harvested and restimulated for 5 h with anti-CD3 (145-2C11, Cat. No. 553057 at 2 µg/ml) and anti-CD28 (clone 37.51, Cat. No. 553294 at 2 µg/ml) antibodies in the presence of 3 µM monensin (BD GolgiStop, Cat No. 554704). The splenocytes were then stained with 0.25 µg of FITC-conjugated rat anti-mouse CD4 (FITC-RM4-5, Cat. No. 553047) and 0.25 µg of PE-conjugated rat anti-mouse IL-4 antibody (PE11B11, Cat. No 554435) by using the Pharmingen staining protocol (left panel). To demonstrate staining specificity, the binding of PE-11B11 was blocked by the preincubation of the conjugated antibody with excess recombinant mouse IL-4 (0.25 µg; Cat. No. 550067) (right panel) or by pre-blocking fixed/permeabilized cells with excess purified 11B11 mAb (5.0 µg; Cat. No. 554433) (data not shown), prior to staining. An approprate isotype control is PE-conjugated R3-34, (Cat. No. 554685). The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified using the cytokine-blocking or mAb blocking controls.
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