Brdu cell proliferation enzyme

A BrdU cell proliferation enzyme-linked immunosorbent assay (ELISA) kit (Calbiochem/Merck Biosciences, Darmstadt, Germany) was used to measure ferroptosis. Tumor cells were treated for 72 h with ART (10 µM) or ART combined with ferrostatin-1 (20 µM), a ferroptosis inhibitor. For more details see “Proliferation” (4.4) as described above.

Cell growth was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay (Roche Diagnostics, Penzberg, Germany). Tumor cells (100 µl, 1×10⁴ cells/ml) were seeded onto 96-well tissue culture plates. After 24, 48 and 72 h, MTT (0.5 mg/ml) was added for an additional 4 h. Thereafter, cells were lysed in a buffer containing 10% SDS in 0.01 M HCl. The plates were then incubated overnight at 37°C, 5% CO₂. Absorbance at 570 nm was measured for each well using a microplate ELISA reader. Each experiment was done in triplicate. After subtracting background absorbance, results were expressed as mean cell number.
Cell proliferation was measured using a BrdU cell proliferation enzyme-linked immunosorbent assay (ELISA) kit (Calbiochem/Merck Biosciences, Darmstadt, Germany). Tumor cells, seeded onto 96-well microtitre plates, were incubated with 20 µl BrdU-labeling solution per well for 8 h, fixed and detected using anti-BrdU mAb according to the manufacturer's instructions. Absorbance was measured at 450 nm.