Cell counting kit 8 cck 8

All of the different kinds of cells and the culture media were provided by the Procell (Wuhan, China). Among them, the A375, A549, H8, U251, 3T3-L1 and PANC-1 cells were cultured in DMEM. The TSSCA and HK-2 cells were cultured in RPMI-1640 medium. All of the cells were supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and streptomycin at 37 °C in 5% CO₂. The cytotoxicity of 6-SH and the other medicines was determined using the Cell Counting Kit-8 (CCK-8, bs-0764P, Bioss, Beijing, China). Additionally, the CompuSyn software was used to calculate the cooperativity index (CI) value, which reflects the synergistic effects of the two medicines.

Primary hepatocytes were isolated from 10 - 12 weeks old female C57BL/6 mice and the isolation was performed as previously described (Liu et al., 2021 (link)). Isolated primary mouse hepatocytes were inoculated at a density of 1 × 10⁴ cells/0.32 cm² growth area per well in rat tail collagen type I-coated 96-well plates for 4 h at 37°C, 5% CO₂, and incubated in a growth medium [William’s E (Gibco, USA), 10% fetal bovine serum, 0.01% dexamethasone, 0.25% insulin and 1% of 100 U/ml penicillin/100 μg/ml streptomycin] for further experiments. Cell Counting Kit-8 (CCK-8) (Bioss, China) assay is used to detect the viability of primary mouse hepatocytes in each group. Briefly, primary mouse hepatocytes were cultured at 37°C and 5% CO₂ for 4 h. The medium was replaced with medium (without serum) and 1% DMSO was used as the control group, while WZB117 was added to the medium at final concentrations of 3.125, 6.25, 12.5, 25, 50 and 100 μmol/L to continue the culture for 48 h. Then 10 μL of CCK-8 solution was added to each mixed well, and after 2 h of continuous incubation, zeroing was completed using blank control wells, and the optical density of each well was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, USA).

The effect of Nec‐1 on synoviocyte viability was assessed using the Cell Counting Kit‐8 (CCK‐8; BIOSS). Briefly, primary synoviocytes were planted into 96‐well plates (10⁴ cells/well), and then stimulated with Nec‐1 (0, 20, 40 and 60 μM for 6, 12 or 24 h). In addition, synoviocytes were subjected to TNF‐α (10 or 100 ng/mL), and simultaneously sensitized with CHX (5 or 10 μg/mL). After discarding the culture, 100 μL DMEM/F12 containing 10% CCK‐8 was added. After 4 h of cell culture at 37°C, synoviocyte viability was checked at 450 nm by a microplate reader (Bio‐Rad).