Meme alpha modification

Renal proximal tubule epithelial cells (RPTEC; Kidney PTEC Control Cells, SA7K Clone, Sigma, Germany, MTOX1030) were cultured on PureCol-coated (Advanced BioMetrix, 5005-B, diluted with 1:30 in HBSS (Sigma H6648), 20 min incubation at 37°C) T75 flasks in MEME alpha Modification (Sigma, M4526) supplemented with RPTEC Complete Supplement (Sigma, MTOXRCSUP), L-glutamine (1.87 mM, Sigma, G7513), gentamicin (28 μg/mL, Sigma, G1397), and amphotericin B (14 ng/mL, Sigma, A2942). Cells were incubated in a humidified incubator (37°, 5% CO 2 ), and every 2-3 days, the medium was changed. At 90-100% confluency, cells were washed with HBSS (Sigma, H6648), detached with accutase (Sigma, A6964), pelleted (140 g, 5 min), and used for seeding in the OrganoPlate. Cells for experiments were used up to passage 3.

To determine the toxic effect of cisplatin on RPTEC tubules in the OrganoPlate, medium of both channels (apical and basal) was replaced at day 6 after seeding with TOX medium (MEME alpha Modification (Sigma, M4526) supplem e n t e d w i t h R P T E C To x S u p p l e m e n t ( S i g m a , MTOXRTSUP), L-glutamine (1.87 mM, Sigma, G7513)) in the presence of 0, 5, 15, 30, 90, 135, or 270 μM cisplatin (Sigma, P4394, stock: 5 mM in 0.9% NaCl (Sigma, S7653) in H 2 0). After 48-h incubation on the rocker platform, phase contrast images were taken and the medium was sampled from the top channel. Samples from in-and outlet were pooled and used for the LDH activity assay. Next, tubes were incubated with WST-8 to determine cell viability. The barrier integrity of the exposed tubules was assessed consecutively of the WST-8 assay. After the exposures and viability measurements, the tubules were fixed with formaldehyde and stained with H2A.X, actin, and ZO-1.