参考图片
Expression of IL-4 by stimulated CD4+ rat splenocytes. Purified splenic CD4+ cells from a 6 month old BN rat were stimulated with plate-bound anti-CD3 (G4.18, Cat. No. 554830 at 25 µg/ml) and soluble anti-rat CD28 (clone JJ319, Cat. No. 554993 at 2 µg/ml) for 2 days in culture together with rat IL-2 (10 ng/ml, Cat. No. 555106) and rat IL-4 (892 ng/ml, Cat. No. 555107), followed by a 3 day incubation with only rat IL-2 and rat IL-4. This was followed by a 6 h stimulation with PMA (1 ng/ml; Sigma, Cat. #P-8139) and Ionomycin (250 ng/ml; Sigma, Cat. #I-0634) in the presence of GolgiStop™ (Cat. No. 554724; aka monensin 2 µM). The cells were then fixed, permeabilized, and subsequently stained with 0.25 µg of PE-mouse anti-rat IL-4 antibody (Cat. No. 555082; far left panel) by using Pharmingen's staining protocol. To demonstrate specificity of staining, the binding of the PE-OX-81 antibody was blocked by preincubation of the PE-conjugated antibody with recombinant rat IL-4 (0.25 µg, Cat. No. 555107; center left panel), and by preincubation of the fixed/permeabilized cells with the unlabeled OX-81 antibody (5.0 µg, Cat. No. 555080; center right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control (far right panel), and verified with the recombinant cytokine blocking (center left panel) and unlabeled antibody blocking (center right panel) specificity controls.
Expression of IL-4 by stimulated CD4+ rat splenocytes. Purified splenic CD4+ cells from a 6 month old BN rat were stimulated with plate-bound anti-CD3 (G4.18, Cat. No. 554830 at 25 µg/ml) and soluble anti-rat CD28 (clone JJ319, Cat. No. 554993 at 2 µg/ml) for 2 days in culture together with rat IL-2 (10 ng/ml, Cat. No. 555106) and rat IL-4 (892 ng/ml, Cat. No. 555107), followed by a 3 day incubation with only rat IL-2 and rat IL-4. This was followed by a 6 h stimulation with PMA (1 ng/ml; Sigma, Cat. #P-8139) and Ionomycin (250 ng/ml; Sigma, Cat. #I-0634) in the presence of GolgiStop™ (Cat. No. 554724; aka monensin 2 µM). The cells were then fixed, permeabilized, and subsequently stained with 0.25 µg of PE-mouse anti-rat IL-4 antibody (Cat. No. 555082; far left panel) by using Pharmingen's staining protocol. To demonstrate specificity of staining, the binding of the PE-OX-81 antibody was blocked by preincubation of the PE-conjugated antibody with recombinant rat IL-4 (0.25 µg, Cat. No. 555107; center left panel), and by preincubation of the fixed/permeabilized cells with the unlabeled OX-81 antibody (5.0 µg, Cat. No. 555080; center right panel) prior to staining. The quadrant markers for the bivariate dot plots were set based on the autofluorescence control (far right panel), and verified with the recombinant cytokine blocking (center left panel) and unlabeled antibody blocking (center right panel) specificity controls.
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