Cck 8 solution

Cells were seeded into 96-well plate (2000–3000 cells/well) with four duplicated wells and cultured for 3, 5 and 7 days. Then 10 μL of CCK-8 solution (Vazyme Biotech, Nanjing, China) per well was added and incubated for 2 h at 37 °C. The absorbance was measured by microplate reader at 450 nm (Molecular Devices, San Jose, CA, USA).

R28 cells with a density of 5 × 10³ cells/well were inoculated in a 96-well cell culture plate and cultivated overnight at 37°C and 5% CO₂. After the treatment with drugs for 24 h, 10 µl CCK-8 solution (Vazyme, Cat. No A311-01/02) was added to each well and the cells were cultivated for another 4 h at 37°C in 5% CO₂. Cell viability evaluation was conducted by measuring absorbance at 450 nm with the use of a Varioskan™ LUX Multi-function microplate reader (Thermo Fisher Scientific, Inc.) [22 (link)].

We prepared exponentially growing cells as single-cell suspensions. We seeded cells in 96-well plates (2,000 cells/well) and assessed them at 0, 24, 48, 72, and 96 h. Time 0 indicated 6 h post-plating. Following the addition of 10 μl of CCK8 solution (Vazyme, Nanjing, China) to each well, we incubated cells for 3 h at 37°C in a 5% CO₂ atmosphere. We incubated the plate with shaking for 5 min. We measured optical density (OD) at 490 nm, using a Microplate reader (Bio-Rad, CA, United States). We performed these experiments in triplicate.

Cell viability was examined by Cell counting kit-8 (CCK-8) assay. HCT116 or HT29 cells (1 × 10³ cells/well) were grown in 96-well plates. After transfection for 24, 48, and 72 h, CCK-8 solution (Vazyme, China) was added to each well by following the kit’s protocol.

A total of 1×10³ transfected cells were seeded in six-well plates and grown at 37°C, 5% CO₂ for two weeks, while the medium was replaced every three days. The colonies were fixed with 4% paraformaldehyde for 30 minutes, stained for 30 minutes with 0.1% crystal violet, and then rinsed with phosphate-buffered saline (PBS; HyClone, Beijing, China), and the number of colonies were counted with a light microscope (MoticAE2000). In 96-well plates, 8×10² transfected cells were seeded and grown at 37°C, 5% CO₂ for six days. Each well received 10 μL of CCK8 solution (Vazyme, Nanjing, China) and 90 μL DMEM medium, and cells were incubated at 37°C, 5% CO₂ for 1 hour. Each well's absorbance was measured at 450 nm at the same time daily, and the results were analyzed using GraphPad Prism Version.

The CCK8 assay was used to evaluate the effect of FA-97 on the viability of SH-SY5Y and PC12 cells. Cells were plated into 96-well plates at a density of 2 × 10⁵ cells/well in medium and cultured overnight. In the preliminary experiment, SH-SY5Y and PC12 cells were treated with H₂O₂ (0, 25, 50, 100, 200, 300, 400, 500, and 600 μM) or FA-97 (0, 0.125, 0.25, 0.5, 1, 2, and 3 μM), respectively. For formal experiments, cells were treated with H₂O₂ (500 μM) and different concentrations of FA-97 (0, 0.25, 0.5, and 1 μM). After 24 h, 20 μl CCK8 solution (cat #A311-01/02, Vazyme Biotech Co., Ltd., Nanjing, China) was added into the medium and incubated for 45 min. The absorbance was measured at 450 nm.