Anti cd3e

Total B cells were isolated from RBC-lysed splenocyte suspensions by positive selection using Miltenyi Biotec MACS LS columns and CD45R (B220) microbeads. B220⁺ or B220⁻ splenocytes were cultured at a final density of 1–2 × 10⁵ cells/mL in complete R10 (RPMI media with 10% FCS plus non-essential amino acids, 1 mM sodium pyruvate and 50 µM 2-β-mercaptoethanol, 20 mM hepes, 2mM L-glutamine, 10units/mL penicillin, 10 µg/mL streptomycin and 20 µg/ml neomycin) at 37 °C, 5% CO₂. For RNA extraction, cells were washed with PBS after 24 h culture and pelleted for later RNA extraction as described below. For proliferation analysis, 10⁶–10⁷ B220⁺ or B220⁻ cells were labeled with 2.5 µM CellTraceViolet (CTV) dye before culturing for 72–96 h prior to analysis by flow cytometry as below. The following stimuli were prepared as indicated in complete R10: anti-IgM, µ-chain specific (Jackson ImmunoResearch; 115-005-075), LPS from Salmonella species (Sigma Aldrich; L7770), HEL (Sigma Aldrich; L6876-5G), anti-CD40 (Biolegend; 102908), anti-CD3e (Biolegend; 100331), anti-CD28 (Biolegend; 122004) and recombinant mouse IL-4 (Biolegend; 715004).

Similar to the coculture system reported by Xu et al. [20 (link)], we stimulated PBMCs with anti-CD3e (2.5 μg/mL, #300432, BioLegend, San Diego, CA, USA), anti-CD28 (1.25 μg/mL) (#302923, BioLegend) and ovarian cancer cell lysate. Briefly, PBMCs were plated at a density of 2 × 10⁶ per well in six-well plates and stimulated with anti-CD3e, anti-CD28, as well as ovarian cancer cell lysate for 3 days. Then CD8⁺ T cells were purified using RosetteSep human CD8⁺ T cell Enrichment Cocktail. For the direct co-culture system, activated CD8⁺ T cells were co-cultured with ovarian cancer cells in 12-well plates at a ratio of 5:1 [21 (link),22 (link)] in the presence of different doses of glutaminase inhibitor 968, with or without anti-PD-L1 antibody. IgG was used as a control. After 24 h, the CD8⁺ T cells were removed with phosphate-buffered saline (PBS), and the attached cancer cells were collected and subjected to flow cytometry. For the indirect co-culture system, transwell cell culture chambers (0.4 μm, CLS3397, Corning, Tewksbury, MA, USA) were used to separate CD8⁺ T cells and cancer cells. CD8⁺ T cells were seeded into the upper chamber, and cancer cells were seeded into the lower chamber at a ratio of 5:1. After exposure to different concentrations of 968 for 24 h, the cancer cells were collected for flow cytometry analysis.