Rpmi 1640

DCs were generated from bone marrow cells of 5- to 7-week-old female BALB/C mice [42 (link)]. Briefly, BM cells were recovered from the femurs and tibias and cultured in a complete culture medium (RPMI 1640 supplemented with 10% fetal bovine serum, all purchased from Cellmax, Sunnyvale, CA, USA) containing 20 ng/mL GM-CSF and 10 ng/mL IL-4 (Peprotech, Cranbury, NJ, USA) for 7 days at 37 °C. Half of the medium was discarded and replaced with an equal-volume fresh medium on the third and sixth days.
Naïve CD4 T cells were isolated from the spleen of WT BALB/C mice and DO11.10 mice (Strain #:003303, purchased from The Jackson Lab, Bar Harbor, ME, USA) using a Naïve CD4 T Cell Isolation Kit (Miltenyi Biotec, Cologne, Germany). The spleens were ground and incubated in a Red Blood Cell Lysis Buffer (Solarbio) for 5 min to release lymphocytes. Magnetic bead sorting operation of naïve CD4 T cells was performed according to the instructions of the isolation kit. The splenic lymphocytes were incubated with a magnetic bead cocktail. The uncaptured cells passing through the column were the target Th cells for the subsequent co-culture.
All animal experiments and handling procedures were approved by the Animal Care and Use Committees of Nanchang University and were performed in accordance with institutional guidelines (Animal ethics code: 2021-0308-035).

Human GBC cell lines EH-GB1 and GBC-SD were purchased from a cell bank (Chinese Academy of Sciences, Shanghai, China). The SGC-996 cell line was provided by Dr. Ying-bin Liu’s lab at Xin Hua Hospital Affiliated with Shanghai Jiao Tong University School of Medicine, China. These cells were maintained in DMEM (EH-GB1 and GBC-SD) and RPMI-1640 (SGC-996) medium containing 10% FBS (cellMax, Beijing, China), penicillin (100 units/ml) and streptomycin (100 μg/ml) in a 5% CO2 humidified incubator at 37 °C (Thermo Scientific).

Human ovarian cancer cell lines CAOV3, OVCAR3, SKOV3, and UWB1.289 were purchased from the American Type Cell Culture. A2780 was purchased from Sigma (Cat# 93112519). HO8910 was purchased from National Collection of Authenticated Cell Cultures of China (Cat# TCHu 24). SKOV3 cells (Cat# HTB-77) were cultured in McCoy’s 5A (BasalMedia, L630KJ). UWB1.289 cells (Cat# CRL-2945) were grown in RPMI 1640 (Cellmax, CGM112.05) and MEGM Bullet Kit (Lonza, CC-3150) at a 1:1 ratio. CAOV3 (Cat# HTB-75) and OVCAR3 (Cat# HTB-161) were cultured in Dulbecco’s Modified Eagle’s Medium (BasalMedia, L110KJ). A2780 and HO8910 were grown in RPMI 1640 (BasalMedia, L210KJ). All culture media contained 10% fetal bovine serum (Gibco, 7471) and 1% penicillin/streptomycin (Invitrogen, 15140-122). The cells were determined to be mycoplasma-free and cultured at 37 °C with 5% CO₂.