Bovine calf serum (bcs)

For in vitro studies, cells from Walker-256 tumour-bearing animals were isolated from the intraperitoneal implant and maintained in culture. Briefly, the ascites fluid from a tumour intraperitoneal implant was collected, and the erythrocytes were lysed with 55 mM NH4Cl, 12 mM NaHCO3, and 0.1 mM EDTA, and the cell suspension was centrifuged at 500 × g, for 5 min, 4 °C. The supernatant was removed, and the pellet containing the tumour cells was seeded in 199 medium (Sigma-Aldrich) supplemented with 10% bovine calf serum (Lonza) and 1% penicillin/streptomycin (Lonza) and maintained at 37 °C and 95% O₂–5% CO₂ atmosphere with 85% relative humidity. Lactate production and glucose consumption in vitro analyses were done as a measurement of the lactate concentration released in the medium, and the glucose consumed was measured accordingly to the manufacturer’s instructions (Bioclin, Brazil). Briefly, cells were cultured in 12-well plates and treated with 50 µM L-leucine (Sigma, USA) for 24 h. The medium was removed and assayed for lactate production^{1 (link)} and glucose consumption^{2 (link)}. Additional Walker-256 cells were seeded in 12-well plate and treated with 50 µM L-leucine (Sigma, USA) for 24 h for mitochondrial respiratory function analyses (Seahorse), protein extraction (western blotting), and RNA extraction (qPCR).

Human embryonic kidney (HEK) 293 Phoenix53 (link) and HeLa (human cervical adenocarcinoma, American Type Cell Culture Collection CCL-2) cells were cultured in Minimum Essential Eagle Medium (Sigma-Aldrich, Austria) supplemented with 10% fetal bovine serum (Lonza), 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin and 1 mM pyruvic acid (sodium salt). NIH/3T3 cells (mouse embryonic fibroblasts, American Type Cell Culture Collection CRL-1658) were cultured in Dulbecco’s Modified Eagle’s Medium (Sigma-Aldrich, Austria) supplemented with 10% bovine calf serum (Lonza), 100 U/ml penicillin and 100 μg/ml streptomycin.
The cells were maintained at 37 °C, 5% CO₂, 95% air and 100% humidity. Subcultures were routinely established every second to third day by seeding the cells into 100 mm diameter Petri dishes following trypsin/ethylenediaminetetraacetic acid (EDTA) treatment. For patch clamp experiments, cells were seeded on glass coverslips (diameter, 10 mm) contained in 30 mm diameter Petri dishes and grown overnight.