BD Horizon™ BV421 Rat Anti-Mouse IL-4

Flow cytometric analysis of IL-4 expressed in activated mouse splenocytes. Splenocytes from C57BL/6 mice were enriched for CD4+ T cells by positive selection using Purified NA/LE Rat Anti-Mouse CD4 antibody-coated plates (GK1.5, Cat. No.553726;10 μg/ml) for 1 hr at 4°C. The CD4+ T cells were harvested and stimulated with plate-bound Purified NA/LE Hamster Anti-Mouse CD3e (145-2C11, Cat. No. 553057;10 μg/ml) and soluble Purified NA/LE Hamster Anti-Mouse CD28 (37.51, Cat. No. 553294; 2 μg/ml) antibody and Recombinant Mouse IL-2 (Cat. No. 550069; 10 ng/ml) and IL-4 (Cat. No. 550067; 50 ng/ml) for 2 days. The cells were expanded in IL-2 and IL-4 for 3 days and then washed and stimulated (4 hr) with PMA (Sigma, Cat. No. P-8139; 5 ng/ml) and ionomycin (Sigma, Cat. No. P-8139; 500 ng) in the presence of BD GolgiPlug™ Protein Transport Inhibitor (Containing Brefeldin A) (Cat. No. 555029). The activated cells were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized using the BD Perm/Wash™ Permeabilization Buffer (Cat. No. 554723) and then stained either with a BD Horizon™ BV421 Rat IgG1, κ Isotype Control (Cat. No. 562868, Left Panel) or with BD Horizon™ BV421 Rat Anti-Mouse IL-4 antibody (Cat. No. 562915, Right Panel). Two-color dot plots showing IL-4 (or Ig isotype control staining) versus autofluorescence were derived from gated events with the forward and light scattering characteristics of intact lymphocytes. Flow cytometry was performed using a BD LSR™ II Flow Cytometry System.

Flow cytometric analysis of IL-4 expressed in activated mouse splenocytes.  Splenocytes from C57BL/6 mice were enriched for CD4+ T cells by positive selection using Purified NA/LE Rat Anti-Mouse CD4 antibody-coated plates (GK1.5, Cat. No.553726;10 μg/ml) for 1 hr at 4°C. The CD4+ T cells were harvested and stimulated with plate-bound Purified NA/LE Hamster Anti-Mouse CD3e (145-2C11, Cat. No. 553057;10 μg/ml) and soluble Purified NA/LE Hamster Anti-Mouse CD28 (37.51, Cat. No. 553294; 2 μg/ml) antibody and Recombinant Mouse IL-2 (Cat. No. 550069; 10 ng/ml) and IL-4 (Cat. No. 550067; 50 ng/ml) for 2 days. The cells were expanded in IL-2 and IL-4 for 3 days and then washed and stimulated (4 hr)  with PMA (Sigma, Cat. No. P-8139; 5 ng/ml) and ionomycin (Sigma, Cat. No. P-8139; 500 ng) in the presence of BD GolgiPlug™ Protein Transport Inhibitor (Containing Brefeldin A) (Cat. No. 555029).  The activated cells were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655), permeabilized using the BD Perm/Wash™ Permeabilization Buffer (Cat. No. 554723) and then stained either with a BD Horizon™ BV421 Rat IgG1, κ Isotype Control (Cat. No. 562868, Left Panel) or with BD Horizon™ BV421 Rat Anti-Mouse IL-4 antibody (Cat. No. 562915, Right Panel). Two-color dot plots showing IL-4 (or Ig isotype control staining) versus autofluorescence were derived from gated events with the forward and light scattering characteristics of intact lymphocytes. Flow cytometry was performed using a BD LSR™ II Flow Cytometry System.