Forma series 2 incubator

NCM460 cells were obtained from INCELL and maintained in M3 base culture medium, supplemented with fetal bovine serum (20%), penicillin (100 U/ml), and streptomycin (100 μg/ml) and incubated at 37 °C in 5% CO₂ incubator as described before. Our selection of the NCM460 cells as the model in the current investigation is based on the fact that they are similar to native human colonic preparations in that they transport TPP and free thiamin via distinct and specific carrier-mediated processes (19 (link), 21 (link), 25 (link), 26 (link)). Also, our findings on the effect of hypoxia were not unique to these cells but were also observed with the human colonic epithelial CCD 841 cells (data not shown). In these studies, we grew the NCM460 cells to 70 to 80% confluency, then maintained them (for the indicated periods) under either a standard normoxic, that is, control (humidified air with 5% CO₂; ThermoFisher Forma Series II incubator) or hypoxic (1% O₂, 94% N₂, and 5% CO₂; Thermo Scientific HERACELL150i incubator) conditions. Chemical hypoxia was induced in NCM460 cells by incubating them in the presence of 250 μM DFO (a hypoxic-memetic agent that chelates iron and prevents the degradation of HIF-1α protein leading to hypoxia; (38 (link), 48 (link))) for 48 h; control cells were run simultaneously and maintained in the absence of DFO.

The A549 cell line (CCL-185™; ATCC, Manassas, VA, USA), from human lung carcinoma epithelium, and the K562 cell line (Rio de Janeiro Cell Bank, BCRJ: 0126, Rio de Janeiro, RJ, Brazil), a human chronic myeloid cell line expressing the BCR-ABL1 protein, were grown in RPMI-1640 culture medium (Merck, Darmstadt, Hesse, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Vitrocell, Campinas, SP, Brazil). WSS-1 [WS-1] (ATCC CRL-2029™) human kidney epithelial cells were grown in high-glucose DMEM (Vitrocell) supplemented with 10% FBS (Vitrocell). All cell lines were maintained at 37 °C under 5% CO₂, in a water jacket CO₂ incubator (Forma Series II incubator, Thermo Fisher Scientific, Waltham, MA, USA).