Expression of IL-3 by stimulated CD4+ Balb/c spleen cells. Purified splenic CD4+ cells from 6 month old BALB/c mice were stimulated with plate-bound anti-CD3 (25 µg/ml final concentration; 145-2C11, Cat. No. 553057) and soluble anti-mouse CD28 (2 µg /ml final concentration; clone 37.51, Cat. No. 553294) for 2 days in culture  together with recombinant mouse IL-2 (10 ng/ml final concentration; Cat. No. 550069) and recombinant mouse IL-4 (0.5 ng/ml final concentration; Cat. No. 550067), followed by a 3 day incubation with only recombinant mouse IL-2 and recombinant mouse IL-4. This was followed by a 5 hour stimulation with plate-bound anti-CD3 (25 µg/ml final concentration) and anti-mouse CD28 (2 µg/ml final concentration) in the presence of GolgiStop™ (2 µM final concentration; Cat. No. 554724). The cells were harvested, stained with 0.06 µg of FITC-conjugated rat anti-mouse CD4 (FITC-RM4-5, Cat. No. 553047), fixed, permeabilized, and subsequently stained with 0.12 µg of PE-conjugated rat anti-mouse IL-3 antibody (PE-MP2-8F8, Cat. No. 554383) by using the BD Pharmingen staining protocol (left panel). To demonstrate specificity of staining, the binding by PE-MP2-8F8 was blocked by each of the following: 1) preincubation of the conjugated antibody with molar excess of recombinant mouse IL-3 (0.12 µg, Cat. No. 554579; center panel) and by 2)   preincubation of the fixed/permeabilized cells with excess unlabeled MP2-8F8 mouse antibody (3 µg; Cat. No. 554380; right panel) prior to staining with the PE-MP2-8F8. The quadrant markers for the bivariate dot plots were set based on the autofluorescence controls and verified using the recombinant cytokine blocking and unlabeled antibody blocking specificity controls.