Pe labeled anti γ δtcr antibodies

To each well of a U bottom 96-well plate 2 × 10⁴ Daudi or Raji tumor cells, 2.5 × 10⁵ expanded γδ T cells, and 5 µg/ml UCHT1, 3.33 µg/ml Fab, 3.33 µg/ml Fab_red, or medium (unstimulated) were given. In addition, the cells were left untreated or treated with different concentrations of AX-024. The plate was incubated at 37°C and 5% CO₂ for 18 h. After incubation, the supernatants were kept at −80°C to quantify the amounts of TNFα and IFNγ (see below). Cells were stained with APC-labeled anti-CD25 (eBioscience) or APC-labeled anti-CD69 (Life Technologies) together with PE-labeled anti-γ/δTCR antibodies (Life Technologies). Cells were analyzed by flow cytometry gating on γδTCR-positive cells.

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.