Detection of mouse IFN-gamma and TNF-a cytokine-producing CD4 T cells using the Attune® Acoustic Focusing Cytometer.
C57BL/6 splenocytes were left unstimulated or stimulated for 5 hours with phorbol myristate acetate (PMA) and ionomycin in the presence of brefeldin A. Cells were then surface-stained with anti-mouse CD4 FITC (Cat. No. MCD0401) and subsequently fixed and permeabilized using the FIX & PERM® Cell Permeabilization Kit (Cat. nos. GAS003 or GAS004) and stained intracellularly with anti-mouse interferon gamma (IFN-gamma) PE-Cy®7 conjugate (Cat. no. A18713) and anti-mouse tumor necrosis factor alpha (TNF-a) Pacific Blue™ conjugate (Cat. no. RM90128). Samples were collected using the Attune® Acoustic Focusing Cytometer (blue/violet) with 488 nm excitation and 530/30 nm bandpass emission filter to detect FITC, and 640 nm longpass filter to detect PE-Cy®7 conjugate. 405 nm excitation and 450/40 nm bandpass emission filter were used to detect the Pacific Blue™ fluorescence. PANEL A: IFN-gamma and TNF-a co-staining of total mouse splenocytes that were left unstimulated (left panel) or stimulated (right panel) with PMA and ionomycin in the presence of brefeldin A. PANEL B: CD4 T cell expression of TNF-a (left panel) and IFN-gamma (right panel) after stimulation with the above stated conditions.