Cell counting kit 8 cck 8 detection kit

The proliferation of A375 and M14 cells was evaluated by Cell Counting Kit‐8 (CCK‐8) detection kit (Beyotime Biotechnology, Shanghai, China). In brief, the transfected cells were seeded at 3 × 10³ cells per 6.4‐mm dish and cultured overnight. At every 24 h, 10 μL CCK‐8 solutions was put in each well, and cells were incubated at 37 °C for another 2 h. To determine the absorbance (A) values of each well, we measured absorbance at 450 nm using a microplate reader (Thermo Fisher Scientific).

Cell proliferation was measured by using Cell Counting Kit-8 (CCK-8) detection kit (Beyotime Biotechnology, China). Cells with a concentration of 5 × 10³ per well were seeded in 96-well plates. To evaluate the cytotoxicity of apatinib on cancer cells, different doses of apatinib were added to the culture. 24, 48, 72, 96 and 120 h later, 10 μL CCK-8 solution was added to each well and followed an incubation at 37 °C for 4 h. The absorbance in each well was measured at 450 nm using a microplate ELISA reader (Bio-Rad Laboratories, CA, USA). The relative cell viability was calculated as follows: relative absorbance = (mean absorbance at each time point/mean respective absorbance at 24 h).

Salvianolic acid B, dihydrotanshinone I, salvianolic acid A, tanshinone II-A, amygdalin, adenosine, cordycepin, schizandrin, schisandrin B, schisantherin A, gypenoside XLIX and kaempferol were purchased from EFEBIO (Shanghai, China¹¹), and their structural information is shown in Supplementary Figure S1. 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) was used as a positive control (Jin et al., 2016 (link)), and obtained from Abcam (Cambridge, United Kingdom). The structures of the above chemicals were unambiguously identified by ¹H NMR and MS spectra, and their purity was demonstrated to be 98% by HPLC-UV. Cell Counting Kit-8 (CCK8) detection kit was obtained from Beyotime (Shanghai, China). LX-2 cells and T6 cells were purchased from Cell Resource Center of Fudan IBS (Shanghai, China). These cells were incubated in Dulbecco minimal essential medium (Sigma, United States) with 10% fetal bovine serum (GIBCO, United States) and penicillin, streptomycin (GIBCO, United States) under a humidified atmosphere with 5% CO₂ at 37°C. The medium was renewed every 2 days.