Apc cy7 anti human cd8

Human peripheral blood mononuclear cells (PBMCs) were isolated from peripheral venous blood using Biocoll (Merck) and Leucosep Tubes (Greiner, Bio-One), following standard protocols. Immune cell composition was analysed using flow cytometry and the following antibodies: FITC anti-human CD3, Pacific Blue™ anti-human CD4, APC/Cy7 anti-human CD8, PE anti-human CD197/CCR7, APC anti-human CD45RA antibody, PerCP/Cy5.5 anti-human CD279/PD-1 (all BioLegend), FITC TCR alpha/beta (eBioscience).
Primary human PBMCs were kept in RPMI 1640 medium supplemented with 10% FBS and 2 mM L-glutamine. Cells were incubated in the absence or presence of plate-bound anti-CD3 (OKT3; BioLegend) and anti-CD28 (CD28.2; BioLegend) antibodies for 120 h. All cells where then stimulated with PMA and ionomycin for 4 h and CD4+ T cells were collected using flow cytometry for RNA (gene expression) and DNA (methylation) analyses.
Some experiments were performed in previously reported genetically modified CD4⁺ Jurkat T cells (22 (link)). Jurkat T cells were kept in RPMI 1640 medium supplemented with 10% FBS and 2 mM l-glutamine. As indicated below, cells were incubated in the absence or presence of plate-bound anti-CD3 (OKT3; BioLegend) and anti-CD28 (CD28.2; BioLegend) antibodies for 24 h. Where indicated, cells were re-stimulated with PMA and ionomycin for 4 h.