Cell counting kit 8 cck 8 assay

For the detection of cell viability, we utilized the Cell Counting Kit‐8 (CCK‐8) assay (GK10001) from GLP Bio (https://www.glpbio.cn/). The assay was performed following the manufacturer's instructions. Transfected cells were cultured in 96‐well plates with 100 µL of culture medium and incubated at 37 °C in a CO₂ incubator for 24 h. Subsequently, 10 µL of CCK‐8 solution was added to each well using a multichannel pipette, and the plates were further incubated for 1–4 h in the incubator. The absorbance of the plate was measured at OD450nm using the H1 Biotek plate reader. Cell viability (%) was calculated using the following formula: Cell viability (%) = [(As‐Ab) / (Ac‐Ab)] × 100, where As represents the absorbance of the experimental wells containing cells, culture medium, CCK‐8, and the tested compound; Ab represents the absorbance of the blank wells containing culture medium and CCK‐8; and Ac represents the absorbance of the control wells containing cells, culture medium, and CCK‐8.

The cell viability of primary human keratinocytes or HaCaT cell lines was determined using the Cell Counting Kit-8 (CCK-8) assay (GLPBIO, United States) according to the manufacturer’s instructions. Briefly, the cells were incubated overnight in 96-well culture plates at a concentration of 1×10³ cells/well and were grown in complete medium for 12–16 h. When 30% confluence was reached, the cells were treated with a mixture of 40 nM RGR siRNA and 5 µl of Lipofectamine 2000 for 48 h, as mentioned previously. Next, 10 µl of CCK-8 solution was added to each well, and the cells were incubated at 37°C for another 2 h. The absorbance at 450 nm was then measured by using a microplate reader.