Rabbit anti phosphozap 70

Peripheral blood mononuclear cells were isolated from healthy donors using a Ficoll–Hypaque gradient. T cells were obtained by negative isolation using the Pan T cell Isolation Kit (Miltenyi Biotech). Cells were taken in RPMI 1640 supplemented with 10% FCS and were rested for 1 h at 37°C in the presence or absence of different concentrations of the inhibitor AX-024 (36 (link)). Cells were left unstimulated or stimulated with 10 µg/ml anti-CD3 antibody UCHT1 for 2 min or 5 min and were fixed with 2% paraformaldehyde for 30 min on ice. Subsequently, cells were permeabilized with 87.7% methanol for 30 min on ice and stained with rabbit anti-phospho-ZAP70 (Cell Signaling) or rabbit phospho-Erk (Cell Signaling) overnight. Next, cells were stained with biotin-labeled anti-CD3 (UCHT1; BioLegend), PE-labeled anti-γ/δTCR antibodies (Life Technologies), and DyLight-labeled anti-rabbit IgG (Thermo Scientific) and subsequently with eFluor 450-labeled Streptavidin (eBioscience). Cells were measured by flow cytometry and gated on CD3- and TCRγδ-positive cells for analysis.