Rpmi glutamax

A375 melanoma cells were purchased from ATCC, MelJuso and IPC298 cells were purchased from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) while the 501Mel melanoma cell line was obtained from Ruth Halaban (Dermatology department, Yale School of Medicine, New Haven, CT, USA). Normal Human Dermal Fibroblasts (NHDFs) were obtained from Heike Hermanns (University Hospital Würzburg, Würzburg, Germany). All melanoma cell lines were cultured in RPMI + Glutamax (Lonza BioWhittaker, Basel, Switzerland) + 10% FBS and 1% PS (10,000 U/mL Penicillin and 10,000 U/mL Streptomycin, Lonza BioWhittaker, Basel, Switzerland) while NHDFs were cultured in DMEM + 10% FBS and 1% PS (10,000 U/mL Penicillin and 10,000 U/mL Streptomycin (PS, Lonza BioWhittaker, Basel, Switzerland). All cells were grown at 37 °C in a humidified atmosphere at 5% CO₂. Cells were regularly tested to be mycoplasma free. Hypoxia treatment was performed at 37°C in a water-saturated atmosphere at 5% CO₂ in a hypoxia station (Invivo₂ 400, Ruskinn Technology Ltd, Bridgend, UK) at 1% O₂.

Human embryonic kidney 293T cells were cultured in 100 mm plates using DMEM with 10% FBS (Lonza) and 1% penicillin/streptomycin (Life Technologies).
Jurkat cells were cultured in RPMI-GlutaMAX (Life Technologies) with 10% FBS (Lonza) and 1% penicillin/streptomycin (Life Technologies).
Peripheral blood mononuclear cells (PBMCs) were isolated from healthy anonymous human buffy coats (provided by the Transfusion Service of Ospedale Maggiore della Carità, Novara, Italy). PBMCs were isolated by Ficoll-Paque PLUS (GE Healthcare) density gradient centrifugation, washed, and resuspended at 2 x 10⁶ cells/ml in RPMI-GlutaMAX containing 10% heat-inactivated FBS (Lonza), 2 mM glutamine, and 100 U/ml of penicillin and streptomycin (Life Technologies). T cells were activated with 1 μg/ml anti-CD3 (clone UCHT1) and anti-CD28 (clone CD28.2) antibodies for 72 hours. Activated T cells were then washed and cultured in the complete medium along with 100 IU/ml rhIL-2 (PeproTech) at 1-2 x 10⁶ cells/ml for ≥ 7 days by changing media every 2–3 days.