Ef670

B16-OVA and B16-OVA-Thy1.1 cells were detached with 2 mmol/L Ethylenediamine tetraacetic acid (EDTA; Gibco) and were prestained with eF450 and eF670 (eBioscience) respectively, following manufacturers’ instructions. The stained cells were then mixed in 1:1 ratio in 96-well round bottom plate (5 × 10⁵ cells per well). Cells were washed three times with FACS buffer (1% FBS in PBS) at 400 × g for 3 minutes at 4°C and incubated with indicated antibodies at 50 μg/mL (50 μL/well) for 30 minutes at 4°C in the dark. Next, the cells were washed three times and were incubated with prewarmed Rabbit Complement (RC; Cedarlane) diluted 1:8 in IMDM complete media (50 μL of RC/well). The cells were incubated for 1 hour at 37°C, after which DNAse (Promega; 1 U/μL) diluted in FACS buffer was added and the cells were washed three times. Finally, the cells were resuspended in 150 μL FACS buffer with 1 mg/mL propidium iodide (PI; Sigma-Aldrich). A total of 100 μL of the stained cells were analyzed on a FACS LSRFortessa (BD) using the software program BD FACSDiva. Further analysis was performed with FlowJo and shown results plotted in GraphPad.

Immature MDCCs conditioned with or without 100 μg/mL cis-UCA were cocultured with allogeneic CD4 T cells stained with proliferation dye EF670 (eBioscience, San Diego, Calif) at a ratio of 1:10 in 96-well plates. CD4 T-cell isolation details are presented in the Methods section in this article's Online Repository. Cells were cocultured at 37°C in a 5% CO₂ humidified atmosphere for 5 days. Anti-CD3/CD28 (1 μg/mL, eBioscience) was added to a portion of CD4 T cells as a positive control. The proliferation of CD4 T cells and the proportion of CD4⁺CD25⁺ forkhead box protein 3 (FoxP3)⁺ CD127⁻ cells were analyzed by using flow cytometry. For details of antibody staining, see the Methods section in this article's Online Repository.

A microscopy-based cytotoxicity assay was established to analyze HER2-CAR T cell function. Briefly, HER2-positive SKBR3 tumor cells were pre-stained with eF670 (Thermo Fischer Scientific, 65-0840-85) for 15 min. Then, 3 × 10⁴ SKBR3 cells were allowed to adhere on 10-mm coverslips in 48-well plates overnight. The next day, control (Ctrl-CAR), HER2-, or antioxidant-enhanced HER2-CAR T cells were untreated or treated with the indicated H₂O₂ concentrations for 1 h. Next, cells were washed and mixed with the indicated ratios of SKBR3 tumor cells in complete RPMI medium and incubated for 8 h and 24 h. Finally, supernatants were collected, aliquoted, and stored at –20°C for further use; remaining adherent cells were fixed, stained with DAPI and phalloidin AF488 (F-actin) (Thermo Fischer Scientific, A12379), and imaged by confocal microscopy as described above. A large scan of 3 x 3 in xy dimensions was acquired using a 20x objective, corresponding to imaging of 9 focal planes per sample. The numbers of cells/focal plane were counted using automated ROI analysis based on DAPI and eF670 signals.
Collected supernatants were used for LDH release assays to assess CAR T cell cytotoxicity toward tumor cells and for CAR T cell cytokine release measurements.