Cck 8 assay kit

NK-92 cells were seeded into 96-well plates (10⁴ cells/well). After 0, 24, 48, and 72 h of culture, cell viability was detected using an CCK8 assay kit (A311-01; Vazyme, Nanjing, China) according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a SpectraMax Absorbance Reader (Molecular Devices, Sunnyvale, CA, USA).

The purchased CCK-8 assay kit (Vazyme, cat#A311–01/02, Nanjing, China) was employed to evaluate the proliferation capacity of cells. Firstly, cells were counted, and 2 × 103 cells were evenly distributed in each well of a 96-well plate. The plate was then placed in a culture incubator and incubated for 24, 48, 72, and 96 h. At the aforementioned time point, freshly prepared CCK-8 reagent was added to the wells, followed by measurement of absorbance and further analysis of the data.

Hepatocellular carcinoma cell lines (HepG2) and normal liver cell lines (THLE3) were cultured as our previous report.^{84 (link)} For lipid accumulation assay of candidate bacteria, cells were seeded in 96-well or 6-well plates containing 100 µL or 2 mL cell growth medium, respectively. When the confluence reached 80%, the medium was replaced with the 90% fresh cell growth medium supplemented with 10% spent culture broth filtrate or BHI medium. After incubation for 24 h, lipid accumulation was evaluated by oil red O staining (Solarbio, Beijing, China) or TG/TC quantification kit (Jiancheng, Nanjing, China) as described previously.^{83 (link)} For the microbial metabolites (tyramine) treatment experiments, cells were seeded in well plates until the confluence reached 80%, and different concentrations of tyramine were treated. The cell viability of tyramine-treatment cells was examined by a CCK8 assay kit (Vazyme, Nanjing, China). After that, the cells were treated with different concentrations of tyramine (0, 5, 25, 50 µM) for 24 h, then the cells were collected for gene and protein expression determination. All experimental conditions were performed in three biological replicates.