Percp conjugated anti human cd3 antibody

Target cells were treated with 50 mg/ml of mitomycin C (Sigma Aldrich) for 30 minutes at 37°C and subsequently washed. 2x10⁵ target cells were seeded in 48-well tissue culture plates and pretreated with 25µM PX-478 for 24 hours under both hypoxic and normoxic conditions before removal of the media. For cell proliferation analysis, mesoCAR T cells and untransduced T cells were stained with 5 mM CFSE at room temperature for 8 minutes. An equal amount of FBS was added to halt the reaction. After washing three times with complete RPMI 1640 medium, CFSE-labeled cells (0.2 × 10⁶/well) were cocultured with either target cells or media, in the absence of exogenous IL‐2, in 48‐well plates, with a final volume of 800 µl/well. After 24 hours, 200 µl of the supernatants were harvested and stored at −80°C. The subsequent cytokine analysis was carried out by enzyme-linked immunosorbent assay (ELISA) to quantify IFN-γ and IL-2. After 72 hours, cells were stained with PerCP-conjugated anti-human CD3 antibody (Clone: HIT3a, BioLegend), and CFSE dilution of CD3+ cells was determined by flow cytometry, as an indicator of proliferation.

The purity of isolated T cells was confirmed using APC-conjugated anti-human CD3 (Clone: UCHT1, BioLegend). PE-conjugated anti-human mesothelin (Clone: #420411, R&D Systems) was used to detect mesothelin expression. FITC-conjugated anti-human CD3 (Clone: HIT3a, BioLegend), PE-conjugated anti-human CD279 (PD-1) (Clone: EH12.2H7, BioLegend), and APC-conjugated anti-human CD366 (Tim-3) (Clone: F38-2E2, BioLegend) antibodies were used to measure the expression of exhaustion markers. For proliferation assays, cells were loaded with CellTrace™ CFSE (Life Technologies, #C34554) according to manufacturer’s instructions, and T cells were detected using PerCP-conjugated anti-human CD3 antibody (Clone: HIT3a, BioLegend). Data were collected using a BD FACSCalibur (BD Biosciences) and analyzed with FlowJo software (v10.6). All assays were performed in duplicate and repeated two to three times.