Edu cell proliferation assay kit

Cell proliferation assay were performed according our previously described, EdU cell proliferation assay kit (Ribobio, Guangzhou, China) was used to detect the effect of BTG1 on cell proliferation [28 (link)]. In a 96-well plate (Guangzhou Jet Bio-Filtration Co., Ltd. Guangzhou, China), 8000 cells were seeded per well. Cellular DNA replication activity was detected by fluorescence microscopy.

Cell proliferation was assessed using EdU Cell Proliferation Assay kit (RiboBio, Guangzhou, China). After different treatments, primary cardiomyocytes were incubated in fresh medium containing 10 μM EdU for 24 h, then cardiomyocytes were washed with PBS, and fixed with 4% paraformaldehyde for 30 min, treated with 0.5% Triton X-100 for 10 min. Cell nuclei were stained with DAPI for 15 minutes. Finally, the proportion of the cells incorporating EdU was determined with fluorescence microscopy.

Cell viability was determined using the CCK-8 Kit (Houston TX, USA) according to the manufacturer’s protocol. In the colony formation test, transfected cells were placed in six-well plates with medium containing 10% FBS. After 14 days, colonies were fixed with methanol and stained with 0.1% crystal violet (Sigma). The stained colonies were counted. Edu assays were performed using the Edu Cell Proliferation Assay Kit (Ribobio, Guangzhou, China) following the manufacturer’s instructions. Then, the percentages of Edu-positive cells were examined in the sample. All experiments were performed in biological triplicates.

BMSCs were seeded in 96-well plates at a concentration of 1 × 10⁵/ml and transfected after incubation for 24 h. At 0, 24, 48, and 72 h after transfection, 10 μl CCK-8 (Dojindo, Japan) reagent was added to each well. After incubation in the dark for 3 h, the optical density (OD) at 450 nm was measured with a microplate reader (Synergy H1, BioTek, USA). Additionally, cell proliferation was visualized with an EdU Cell Proliferation Assay kit (RiboBio) and a fluorescence microscope (Nikon, Japan).

The MTT assay was used to detect the cell growth rate. Briefly, cells were plated in 96-well plates (4 x 10³ per well) and received different concentrations of drug treatment.
The detailed procedures have been described by Dou et al. (2016) (link). The colony formation assay, cells were cultured in 24-well plates (800 cells/well) and treated with different treatments. After 2 weeks, the 4% paraformaldehyde was adopted for fixation and crystal violet was used for dyeing. The visible colonies were captured by Molecular Imager Gel Do XR + System (Bio-Rad, Hercules, CA, USA) and calculated applying ImageJ software (National Institutes of Health, Bethesda, MD, USA). The detailed procedures have been previously described by Wang et al. (2011) (link). The 5-ethynyl-20-deoxyuridine (EdU) labeling analysis was carried out in 24-well plates (2 x 10⁴ cells) using the EdU cell Proliferation Assay Kit (Ribobio, Guangzhou, China). After different concentrations of drug treatment, 10 μmol/L EdU was added to the cells, and the cells were incubated for 12 h at 37°C. Cells were fixed with 4% paraformaldehyde. Then DAPI was utilized for nuclear staining and fluorescence microscopy was applied for imaging.

The EdU Cell Proliferation Assay Kit (RiboBio, Guangzhou, China) was used to measure cell proliferation. C2C12 myoblasts or HUVECs, which were cultured in the conditioned medium with different concentrations of EVs or SKP-SCs, were incubated in a fresh medium containing 50 μM 5-ethynyl-2′-deoxyuridine (EdU) and cultured in a cell incubator at 37 °C for 2 h. After rinsing in PBS, the cells were fixed in 4% paraformaldehyde for 30 min and treated with 0.5% Triton X-100 for 10 min. Hoechst 3342 was used for staining the nuclei for 15 min. Finally, the proportion of C2C12 myoblasts or HUVECs that proliferated in the culture medium containing EdU was counted.