Cell counting kit 8 cck 8 reagent

The Cell Counting Kit‐8 (CCK‐8) assay was used to measure cell viability. BMDMs were cultured in a 96‐well plate (5 × 10⁴ cells/well) with 100 µL of 1640 medium in each well treated with different concentrations of erastin or RSL3 and grown in a 37 °C constant temperature incubator containing 5% CO₂. Cell Counting Kit‐8 (CCK‐8) reagent (20 µL, MedChemExpress, New Jersey, USA) was added to each well according to the manufacturer's instructions and incubated for 2 h prior to measuring the OD450 nm with a microplate reader (Bio‐Rad, Berkeley, USA). Each group was replicated with three wells and each experiment was repeated at least thrice.
Then the BMDMs were cultured in Hepa1‐6 CM or normal medium for 48 h and then plated in a 6‐well plate (1 × 10⁶ cells/well) and treated with 2 µm erastin or 40 nm RSL3 with or without 4 µm ferrostatin‐1 (Fer‐1) for 0–48 h, followed by the count of living cells using a Countess 3 FL automated cell counter (Lot#AMQAX2000, ThermoFisher, USA). And the LIVE/DEAD Viability/Cytotoxicity Kit (Lot#1 976 809, Invitrogen, CA, USA) was also used to detect the number of living dead cells.

Cells were seeded in 96-well plates at a density of approximately 4000 cells/100 µL per well and incubated for 24 h. Subsequently, they were exposed to different concentrations of 4-OH tamoxifen (0, 2, 4, 8, 16, 32 μM) for 72 h. Cell viability was determined using Cell Counting Kit-8 (CCK-8) reagent (MedChemExpress, Shanghai, China). Following a 2-h incubation, the absorbance was measured at 450 nm. The ratio of the absorbance corresponding to each drug concentration to the absorbance without drug addition is the cell viability under the influence of 4-OH tamoxifen. p < 0.05 was considered a statistically significant difference in drug sensitivity.