Microplate analyzer

Primary mice myocardial fibroblasts were cultured in DMEM with 10% fetal bovine serum (GIBCO, Carlsbad, CA, USA) at 37°C with 5% CO₂. To investigate the role of TGF-β1, the fibroblasts were exposed to TGF-β1 for 6, 16, 24, 36, and 72 h sequentially at 0, 2.5, 5, 10, and 15 μg/ml, respectively. After the TGF-β1-stimulated myocardial fibroblast injury model was successfully obtained, the cells were treated with TSF for 72 h at the concentrations of 100 and 250 μg/ml.
An MTT assay was used to detect whether TSF had an effect on cell viability. Being starved overnight with DMEM/F12 medium without fetal bovine serum (FBS), C57BL/6J mouse primary myocardial fibroblasts were then grown in 96-well plates and incubated with TSF at the concentrations of 0, 100, 250, 500, 750, and 1,000 μg/ml for 72 h. Each well was added with 1/10 volume of MTT solution and incubated at 37°C with 5% CO₂ for 4 h. After adding formazan lysate, the assay was performed at 490 nm wavelength with a microplate analyzer (Thermo Fisher Scientific, Waltham, MA, USA). The experimental methods were completed according to the kit instructions, and cell viability was calculated.

The cells with inducible gene expression were inoculated for successful attachment. After the rinse with PBS, MTT was added and incubated subsequently at 37°C for 4 h. Then, DMSO was added and vibrated gently to make the complex dissolve sufficiently. Finally, a microplate analyzer (Thermo Scientific, USA) was used to measure the absorbance of crystalline complex. For the analysis of cell proliferation, it was expressed as the average optical density at 490 nm ± SEM (n = 3).

Transfected cells were seeded into 96-well plates at the density of 1 × 104 cells/well, and incubated for 24, 48, and 72 h. Ten microlitres of CCK-8 solution (Yeasen, China) was added to each well and then incubated for 2 h. The medium was then removed and the plates were washed twice. The optical density was measured at 450 nm using a microplate analyzer (Thermo Fisher, CA, USA).