Cell count kit 8

Cell Count Kit-8 (CCK-8, Beyotime, Beijing, China) was used to measure the number of viable cells from different treatments. Briefly, cells were digested, resuspended and reseeded into 96-well plates. Then, 10% CCK-8 solution was added into each well. After 1-h incubation, the absorbance was determined on ELISA reader (Bio–Rad, Hercules, CA, U.S.A.) at a wavelength of 450 nm.

The cell count kit-8 (CCK, Beyotime, China) was used to quantitatively identify the cellular viability. The cultured CCs were rinsed twice with PBS, and then DMEM/F12 with 10% CCK-8 was added to these samples in a separate volume of 0.2 mL. After incubation for one hour, the absorbance was measured at 450 nm under a microplate reader (Bio-TEK, USA). Four parallel replicates of each sample at each time point were prepared during this cell viability assay.

Gambogic acid was purchased from Sigma-Aldrich (St. Louis, MO, USA). Cy3-labeled siRNA (XIAP siRNA) was purchased from RiboBio (Guangzhou, China) with the following sequences: XIAP siRNA (sense): 5-AAG UGG UAG UCC UGU UUC AGC-3. Monoclonal antibodies (XIAP) and HRP-linked antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Cell Count Kit-8, 4′,6-diamidino-2-phenylindole (DPAI), and LysoTracker Green were from the Beyotime Institute of Biotechnology (Shanghai, China). Annexin V-FITC Apoptosis Detection Kit and Cell Cycle and Apoptosis Detection Kit were from Beijing Biotopped Technology Co., Ltd. (Beijing, China). Tween-80, squalene, and glycerol were manufactured by Shanghai Energy Chemical Co., Ltd. Immunohistochemistry Kit (D601037-0050) was purchased from Sangon Biotech (Shanghai, China). Deionized water was produced in the laboratory. The reagent-grade chemicals used in the tests were all used exactly as provided to the researchers.

To evaluate the cytotoxicity of compounds, a series of diluted concentrations of compounds were incubated with Vero E6 or Calu-3 cells in a 96-well plate (1 × 10⁴ cells/well) for 24 h, following cell viability assessment with a cell count kit-8 (Beyotime, Shanghai, China, #C0039) according to the manufacturer’s instructions. The OD₄₅₀ values of the compound treated cells (OD_compound), DMSO-treated cells (OD_DMSO), and a medium without cells (OD_blank) were obtained for calculating the normalized cytotoxicity under each concentration, which was expressed as: % cytotoxicity = 100% − (OD_compound − OD_blank) / (OD_DMSO − OD_blank) × 100%. The CC₅₀ for each compound on each cell type was calculated using Graphpad Prism 8.0 software.

Cells were seeded overnight at 1 × 10³ cells/well in 96-well plates, and 10 µL of Cell Count Kit-8 solution (Beyotime Biotechnology, Jiangsu, China) was applied to each well and incubated for 2 h at 37 °C. Cell survival was assessed at the end of each time point (0, 24, 48, 72 h). At 540 nm after incubation, absorbance was measured.

Quantitative evaluation of cell viability was performed using a cell count kit-8 (Beyotime), according to the manufacturer's instructions. The absorbance at 450 nm was determined using a microplate reader.