Cell counting kit 8 (cck8)

The IPEC-J2 cells grown at logarithmic phase were treated with 0.1% trypsin to prepare a single cell suspension and then seeded in a 96-well cell culture plate. The number of seeding was 1 × 10⁴ cells/well and cultivated for 24 h in an incubator (37°C) with constant concentration of CO₂ (5%). Then, the medium was discarded, the wells were washed with sterile PBS, added with medium containing 0, 1, 5, 10, 50, 100, 200, 500, 800, and 1,000 μM/L H₂O₂, respectively, and cultured for 24 h (six replicate wells per group). For each well, the culture medium was discarded, added with 100 μl of medium containing 10% Cell Counting Kit-8 (Biosharp, China), and incubated for 2 h at 37°C. The OD₄₅₀ value was measured with a microplate reader. The concentration of H₂O₂ applied in the subsequent experiments was determined based on its reduction of cell viability by 50% with the cells treated with H₂O₂ for 24 h to establish a cellular oxidative stress model. For the treatment of Z. Tk, the cells were treated with the medium containing 1 × 10⁻², 5 × 10⁻³, 1 × 10⁻³, 5 × 10⁻⁴, 1 × 10⁻⁴, and 5 × 10⁻⁵ dilutions of Z. Tk extracts for 3 h, then added 100 μl of medium containing 10% Cell Counting Kit-8 (Biosharp, China), incubated at 37°C for 2 h, and the OD₄₅₀ values were measured with a microplate reader.

CCK-8 assays were performed to detect cell viability using the Cell Counting Kit-8 (BS350-B, Biosharp, Hefei, China). The primary RGCs and Müller cells were seeded onto 96-well plates for 24 h. Then, the required dose of drugs or H₂O₂ were added. After 24 h culture, 10 μL of CCK-8 reagent were incubated in each well at 37 °C for 1 h. The absorbance value was detected using the Filter Max F5 Microplate Reader (Molecular Devices, San Jose, CA, USA) at 450 nm wavelength.