Cell counting kit 8 assay

C2C12 cells at 70% confluence were selected and prepared into a single‐cell suspension. To detect the effect of Tent5a knockdown on myoblast viability, C2C12 cells were inoculated in 96‐well plates, and 2000 cells in 100 µl of growth medium were seeded in each well. After incubation for 24 h, the Cell Counting Kit‐8 assay (YZ‐CK04, Solarbio) was performed according to the manufacturer's protocol. The wound‐healing assay was used to quantify cell migration. Cells were inoculated with a 6‐well plate and scratched when the cell density increased close to 100%. The observation time points of the wound‐healing assay included 0, 24, and 48 h. To detect the effect of Tent5a knockdown on myoblast apoptosis, C2C12 cell apoptosis was induced by H₂O_2, and a TUNEL assay (A113, Vazyme) was conducted according to the manufacturer's protocol. After culturing in differentiation medium for 5 days, C2C12 cells were fused and formed mature myotubes. Immunofluorescence staining and qPCR were used to detect the effect of Tent5a knockdown on myoblast differentiation.

Cell Counting Kit‐8 assay (CCK 8, Solarbio, China) was used to evaluate the effect of ethanol (100 mM) and alda‐1 (20 μM) on BMSCs and HUVECs. In brief, 5 × 10³ cells in 100 μl medium per well were seeded in 96‐well plates. According to the protocol, every well was added with 10 μl CCK 8 solution and 90 μl medium. Then the absorbance values of supernatants were tested at 450 nm after incubation of the plates in 37°C for 2 h.

The effect of rTsDPP1 on RAW264.7 macrophage cellular viability was determined using a Cell Counting Kit-8 assay (CCK-8; Solarbio, Beijing, China) [33 (link), 34 (link)]. Briefly, the cells were seeded in a 96-well plate (approximately 1 × 10³ cells per well) and cultured under the same conditions as described above. rTsDPP1, recombinant thioredoxin (TRX) tag protein and AW ESA were added into the medium and cocultured with cells for 48 h. Then, 10 µL CCK-8 solutions were added to each well and incubated for another 2 h. The absorbance at 450 nm was measured using a multimode reader (SpectraMax i3X; Molecular Devices, USA). Cell viability was presented as the cell survival rate according to the following formulas: cell survival rate = (OD values of test group – OD values of blank control)/(OD values of PBS control group – OD values of blank control) × 100%.

Cell proliferation was also analysed with the Cell Counting Kit (CCK)-8 assay (Solarbio, Beijing). A375 cells were collected, washed, resuspended, counted using a haemocytometer, added to 96-well plates at 2000 cells/well and cultured at 37 °C and 5% CO₂. Cell proliferation was analysed every day for 4 days. Briefly, 10 μg CCK-8 solution (5 mg/ml) was added to the cells (10 μl/well), followed by incubation for 2 h. Finally, absorbance at 450 nm was measured with a microplate reader (DeTie, Nanjing, China).