Edu apollo in vitro imaging kit

For the analysis of cell proliferation stages, cells were cultured and seeded into a 12-well culture plate and transfected for 48 h. After pre-cooling PBS washing, cells were fixed in 70% ethanol and stored at −20 °C overnight. Then, a Cell Cycle Analysis Kit (Thermo Fisher Scientific, MA, USA) was used in flow cytometry analysis of the cell cycle. A BD Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA) was utilized for the analysis of myoblasts. The FlowJo software (7.6, Tree Star, Ashland, OR, USA) was used for data processing.
For the EdU assay, myoblasts were seeded into a 12-well culture plate and transfected when the cells density reached 70–80%. After 48 h transfection, cells were fixed by 4% formaldehyde for 30 min and stained by using EdU Apollo In vitro Imaging Kit (C10310, RiboBio, Guangzhou, China). A fluorescence microscope was utilized to acquire images, and ImageJ software was used for the analysis of data.
For the CCK-8 assay, primary myoblasts were seeded into 96-well culture plates and transfected. Then, the cell growth condition was monitored at 12, 24, 36, and 48 h by using the TransDetect CCK Kit (TransGen Biotech, Beijing, China) following the instruction book. The absorption spectra at 450 nm were detected by using an iMark microplate absorbance reader (Bio-Rad, Hercules, CA, USA). All data were acquired from six independent repeats.

The cells were cultured until 50% density and transfected with the miR-24-3p mimic, the mimic NC, the ACVRIB siRNA (si-ACVR1B), and the siRNA negative control (si-NC). The cells were subjected to the 5-Ethynyl-2’-deoxyuridine (EdU) assay 24 h after transfection using an EdU Apollo In Vitro Imaging Kit (RiboBio, Guangzhou, China). We captured three randomly selected fields using a fluorescence microscope (TCS SP8, Leica) and determined the number of EdU-stained cells. ImageJ was used to determine the percentage of EdU-positive cells.

For the EdU assay, we planted DPCs using a 24-well cell culture dish. After the DPCDPC reached 50% density, cells were transfected. After 2 days, we used 4% paraformaldehyde (Solarbio, Beijing, China) to fix DPCs for 30 min. Then, we used Triton X-100 to make cell membrane permeabilization. Finally, we treated DPCs using an EdU Apollo In Vitro Imaging Kit (RiboBio, Guangzhou, China). After the DPCs were dyed, we used a fluorescence-inverted microscope (DMi8, Leica, Germany) to acquire images of stained cells. Three regions were randomly selected for evaluating the number of stained cells, and Image Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA) was used for data analysis.

A 24-well cell culture dish was used to culture the SMSCs. After the cells’ density reached 40%, we transfected miR-181a mimic and mimic NC into the SMSCs. After 48 h of transfection, we incubated the cells with EDU for two hours. Then, we used 1X PBS (Solarbio, Beijing, China) to wash the cells. Next, we used 4% paraformaldehyde (Solarbio, Beijing, China) to fix SMSCs for 0.5 h. Finally, we used EdU Apollo In Vitro Imaging Kit (RiboBio, Guangzhou, China) to treat the SMSCs. A fluorescence inverted microscope (Nikon, Tokyo, Japan) was used for cell image acquisition. Image-Pro software was used for data analysis. In each group of treatments, three areas were randomly selected to calculate the number of stained cells.

H292, A549, and Hcc827 cells were seeded in 96-well plates (5 × 10³ cells per well) cultured with normal medium, CAF CM, or senescence-like CAF CM for 48 hours, respectively. Cell proliferation was determined by the EdU Apollo in Vitro Imaging Kit (RiboBio) according to the manufacturer’s protocol, and cells were exposed to EdU before staining for 2 hours.