Canto 2

For annexin V-PI assays, cells were stained with annexin V-fluorescein isothiocyanate (FITC) and PI and evaluated for apoptosis/necrosis by flow cytometry according to the manufacturer’s protocol (BD Bioscience). H₂O₂ was diluted in phosphate-buffered saline (PBS), added to the medium at 200 μM, and immediately added to the cells for a 90-min treatment. The cells were then washed in PBS, and fresh medium was added. After 8 h, the cells were trypsinized and recombined with cells floating in the medium. The collected cells were washed with cold PBS, stained with annexin V-FITC and PI in binding buffer for 15 min at room temperature in the dark, and analyzed using Canto II (Becton, Dickinson Biosciences). Cell populations were determined using FlowJo software (Tree Star Software, San Carlos, CA). The quadrants were chosen using unstained and single-stained control samples.
To quantify the level of reactive oxygen species, cells were washed briefly with warm PBS and incubated with 5 μM 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) (Invitrogen; C400) in PBS for 20 min. The cells were then quickly washed and allowed 20 min of recovery in MEM supplemented with 10% FBS. Cells were harvested with trypsin, resuspended in 0.5% bovine serum albumin (BSA) in PBS, and analyzed using Canto II (Becton, Dickinson Biosciences). Cell populations were determined using FlowJo software.

Human tumor cell line expression of antigens and receptors was characterized using FACS analysis utilizing the Canto II (Becton Dickinson, Franklin Lakes, USA) platform. Cells were quantified and isolated from culture at 5x10⁵-1x10⁶ cells per FACS tube, stained with anti-CD19 FITC (Miltenyi Biotec Cat#130-113-645; RRID:AB_2726198), anti-CD80 BV421 (BioLegend Cat#305222; RRID:AB_2564407) or anti-CD80 PE (ImmunoTools Cat#21270804; RRID:AB_2923118) and anti-CD86 APC (ImmunoTools Cat#21480866; RRID:AB_2923116), washed twice each before and after antibody application with PBS and incubated at 4°C for 30 min before analysis.
Murine tumor cell line and primary cell expression of antigens was evaluated on the MACSQuant X (Miltenyi Biotec, Bergisch Gladbach, Germany) and Canto II (Becton Dickinson, Franklin Lakes, USA) platforms using anti-mouse CD19 (1D3) APC (ImmunoTools Cat#22270196X2; RRID requested) or anti-mouse CD19 BV510 (6D5) (BioLegend Cat#115545; RRID:AB_2562136), anti-mouse CD80 (16-10A1) PE (BioLegend Cat#104707; RRID:AB_313128) and anti-mouse CD86 (GL-1) PE/Cy7 (BioLegend Cat#105014; RRID:AB_439783) antibodies.

Isolated T cells were collected by centrifugation (330 × g, 5 min at 4 ℃) and cell counts were determined by using a Countess Counter (Invitrogen). For surface staining, cells were resuspended in PBS (with 2% FBS) containing the antibody, incubated on ice for 30 min, washed in PBS (with 2% FBS), and analyzed using flow cytometry. Flow cytometry assay was performed on CANTO II (BD Biosciences). For intracellular molecule detection, fixation and permeabilization buffers (eBioscience) were used according to the instructions. Data were collected on BD CANTO II (BD Biosciences) with FACSCanto software version 2.1 (BD). Antibodies used were described in Table S2.