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Flow cytometric analysis of TNF expression in stimulated human peripheral blood lymphocytes. Human peripheral blood mononuclea r cells were stimulated for 6 hours with Phorbol 12-Myristate 13-Acetate (Sigma P-8139; 50 ng/ml final concentration) and Ionomycin (Sigma I-0634; 1 λg/ml final concentration) in the presence of BD GolgiStop™ Protein Transport Inhibitor (containing Monen sin) (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 either BD Horizon™ BUV395 Mouse IgG1 κ Isotype Control ( Cat. No. 563547; Left Panel) or BD Horizon™ BUV395 Mouse Anti-Human TNF antibody (Cat. No. 563996; Right Panel). Two-color flow cytometric dot plots show the correlated expression patterns of TNF (or Ig Isotype control staining) versus Autofluorescence 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.
Flow cytometric analysis of TNF expression in stimulated human peripheral blood lymphocytes. Human peripheral blood mononuclear cells were stimulated for 6 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 either BD Horizon™ BUV395 Mouse IgG1 κ Isotype Control (Cat. No. 563547; Left Panel) or BD Horizon™ BUV395 Mouse Anti-Human TNF antibody (Cat. No. 563996; Right Panel). Two-color flow cytometric dot plots show the correlated expression patterns of TNF (or Ig Isotype control staining) versus Autofluorescence 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.