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Two-color flow cytometric analysis of IL-17A expression in stimulated human peripheral blood lymphocytes. Human peripheral blood mononuclear cells were stimulated for 5 hours with Phorbol 12-Myristate 13-Acetate (Sigma P-8139; 50 ng/ml final concentration) and Ionomycin (Sigma I-0634; 1 μg/ml final concentration) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monensin) (Cat. No. 554724). The cells were harvested, washed with BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656), and fixed and permeabilized with BD Cytofix/Cytoperm™ Fixation and Permeabilization Solution (Cat. No. 554722). The cells were then washed and stained in BD Perm/Wash™ Buffer (Cat. No. 554723) with APC Mouse Anti-Human CD4 antibody (Cat. No. 555349/561840/561841) and either BD Horizon™ BV650 Mouse IgG1 κ Isotype Control (Cat. No. 563231; Left Panel) or BD Horizon™ BV650 Mouse Anti-Human IL-17A antibody (Cat. No. 563746; Right Panel). Two-color flow cytometric dot plots show the correlated expression patterns of IL-17A (or Ig Isotype control staining) versus CD4 for gated events with the forward and side light-scatter characteristics of intact lymphocytes. Flow cytometric analysis was performed using a BD™ LSR II Flow Cytometer System.