Glutamax

Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells were transfected to stably express CTLA-4 and/or OX40. The following cell lines were generated and used: CHO-CTLA-4 (0.1 × 10⁶ receptors/cell), HEK-CTLA-4 (0.8 × 10⁶ receptors/cell), CHO-CTLA-4-OX40 (0.7 × 10⁶ CTLA-4 receptors/cell; 4.8 × 10⁶ OX40 receptors/cell) and CHO-OX40 (1.1 × 10⁶ receptors/cell). Moreover, CHO-FcγRI (0.1 × 10⁶ receptors/cell) were used for crosslinking of ATOR-1015. All cell lines were cultured in RPMI-1640 with Glutamax (Gibco) supplemented with 10% fetal bovine serum (FBS, GE Healthcare), 10 mM HEPES (Gibco), zeocin (250 μg/ml) and/or geneticin (600 μg/ml, Gibco) depending on selection pressure.
MB49 bladder cancer cells (EMD Millipore) were cultured in modified DMEM (high glucose, Glutamax and sodium pyruvate) supplemented with 10% FBS. MC38 colon carcinoma cells (Kerafast) were cultured in RPMI-1640 with Glutamax supplemented with 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate and 0.05 mM 2-mercaptoethanol.

The human osteosarcoma cell lines U2OS-TrkA/p75-SHC1 and U2OS-TrkB/p75-SHC1 were acquired from Eurofins DiscoverX Corporation (Fremont, CA, USA). Cells were maintained subconfluent in MEM medium supplemented with 10% FBS, 1% penicillin/streptomycin, and hygromycin/geneticin/puromycin. Immortalized SH-SY5Y cell line expressing TrkB was purchased from Kerafast (Boston, MA, USA) and maintained in complete growth medium, consisting of RPMI1640, GlutaMAX, 10% FBS, 1% penicillin/streptomycin, and 0.3 mg/mL geneticin. The PC12 cell line was purchased from ATCC (LGC Standards GmbH, Wesel, Germany) and maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. All cell cultures were maintained at 37 °C in a humidified atmosphere with 5% CO₂.