Cck 8 kit

The proliferative capacity of HCCLM3 with/without TAF6 downregulation was observed by CCK-8 assay and EdU assay. Three group cells were seeded in 96-well plates at 3×10³ cells per well for the CCK-8 assay. Cell proliferation was detected using a CCK8 kit(Bioss, Beijing, China). 10μl CCK-8 reagent was added to each well at 0h, 24h, 48h, and 72h after transfection, and the cells were incubated at 37°C for 1.5h without light. The absorbance was measured at 450nm and we repeated each experiment at least three times. In addition, three group cells were seeded in 96-well plates at 1.5×10⁴ cells per well for EdU assay. According to the instructions of the YF^®594 Click-iT EDU(UE, Shanghai, China) staining kit, the EDU was diluted to 50 μmol/L by the complete medium. Then we added 100 μL to each well and incubated for 2 h. Next, the medium was removed, and the cells were fixed in 4% paraformaldehyde and neutralized with 2mg/mL glycine solution, and washed twice with 3%BSA. 0.5%Triton X-100 was used as the osmotic enhancer, and the required Click-iT working solution was configured and incubated for 30min under dark conditions.1×Hoechst 33342 solutions were used for Nuclear redyeing. Finally, Images were taken with a fluorescence microscope and analyzed with Image J(version1.8).

Cell proliferation was detected using a CCK-8 Kit (Bioss, China). Logarithmically growing cells were collected. After counting, the cell suspension concentration was adjusted, and the cells were inoculated into a 96-well plate at a density of 2 × 10³ cells/well. According to the manufacturer’s instructions, 10 µL CCK-8 solution was added, and the cells were incubated for 2 h. The absorbance at 450 nm was measured using an RT-6000 microplate reader (Rayto, China). Cell proliferation was expressed as the cell survival rate. The calculation formula was as follows: cell survival rate (%) = (OD value (sample) - OD value (PBS)) / (OD value (control group) - OD value (PBS)) × 100%.

For the in vitro cell proliferation assay, the CCK-8 assay was performed using the CCK-8 kit (Bioss, Beijing, China) according to the manufacturer’s protocol. An enzyme immunoassay instrument (Thermo Fisher Scientific, USA) was used to detect the OD value at 450 nm. An in vitro colony formation assay was performed to determine whether colony formation was possible in vitro, and the procedure was performed as previously described [68 (link), 69 (link)]. Finally, the migration ability of the cells was determined using the Transwell chamber system (BD Biosciences, BD Biosciences, Franklin Lakes, NJ, USA).

The cell proliferation ability of RA‐FLS was measured by using CCK8 kit (BA00208; BIOSS). Initially, 3 × 10⁴ RA‐FLSs were inoculated in each well and incubated to an extent of 70–90% fusion. The RA‐FLS in the logarithmic growth phase were thereafter grouped and transfected. Three different repeat wells were set for each group and incubated for 0, 12, 24, 48, and 72 h, respectively. At each of the above experimental time points, 10 μl CCK‐8 solution was added into each well and incubated for 1.5 h at 37℃. Finally, the absorbance value of each well was measured at 450 nm.

GH₃ proliferation was assessed by the cell counting kit-8 (CCK-8) method, 5-ethynyl-2′ deoxy uridine (EdU) incorporation assay and proliferating cell nuclear antigen (PCNA) expression. Firstly, the rate of GH₃ proliferation was determined with the CCK-8 kit (Bioss, Beijing, China) according to the manufacturer’s instructions. The number of viable cells was assessed by measuring the absorbance at 450 nm using a Synergy 2 Multi-Mode Reader (Bio Tek Instruments, Inc., Winooski, VT, USA). Secondly, DNA synthesis was examined with EdU incorporation assay (YF^® 555 Click-iT EdU Imaging Kit, Suzhou US EVERBRIGHT, Suzhou, China) to evaluate GH₃ proliferation. The EdU positive cells were counted and normalized by the total number of Hoechst 33,342 stained cells. Lastly, GH₃ proliferation was evaluated by PCNA expression, which is the auxiliary component of DNA polymerase δ and constitutes a useful proliferation marker.