Multifunctional enzyme marking instrument

To assess the cytotoxicity of PAB, human normal hepatocyte (HL-7702 cell line) and renal cell line (HK-2 cell) were seeded into 96-well culture plates (5 × 10³ cells per well) and 24-well culture plates (2 × 10⁴ cells per well) with complete culture media, and followed by incubation at 37°C and 5% CO₂ for 6 h. PAB and ABZ-SO at a final concentration of 80, 40, 20, 10, 5, 2, 1, 0.5, 0.2 and 0 μg/ml were added into the cell culture plates, respectively (n = 6). After treatment with PAB and ABZ-SO for 48 h, cell counting kit-8 (CCK-8) solution (10 μl per well) was added into 96-well culture plates to continuously incubate for 2 h, and the optical density was read under a multifunctional enzyme marking instrument (BioTek, US), and then cell viability was calculated using the following formula: cell viability = [(OD_treatment − OD_blank)/(OD_control − OD_blank)] × 100%, and IC50 values were calculated using the Graphpad/Prism software. In addition, cell morphologic alterations in 24-well culture plates were observed after staining with 1% crystal violet (Solarbio Co., Ltd., Beijing, China) under the indicated inverted microscope. These experimental processes were conducted with three independent biological replicates following the manufacturer’s specifications (Beyotime Biotechnology Co., Ltd., Shanghai, China).