Expression of GM-CSF 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; clone 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 (1 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 hours stimulation with plate-bound anti-CD3 (25 µg/ml final concentration) and anti-mouse CD28 (2 µg/ml final concentration) in the presence of BD GolgiStop™ (2 µM final concentration; Cat. No. 554724). The cells were then stained with 0.06 µg of FITC rat anti-mouse CD4 (FITC-RM4-5, Cat. No. 553047) fixed, permeabilized, and subsequently stained with 0.12 µg of PE rat anti-mouse GM-CSF antibody by using the Pharmingen staining protocol (left panel). To demonstrate specificity of staining, the binding of PE-MP1-22E9 was blocked by the preincubation of the conjugated antibody with recombinant mouse GM-CSF (1 µg, Cat. No. 554586; center panel), and by preincubation of the fixed/permeabilized cells with the unlabelled MP1-22E9 mouse antibody (10 µg, Cat. No. 556916; right panel). The quadrant markers for the bivariate dot plots were set based on the autofluorescence control, and verified with the recombinant cytokine blocking (center panel) and unlabelled antibody blocking (right panel) specificity controls.