Enzyme linked immunoassay

ADSCs (Passage 3, 1 × 10³ cells/well) were seeded in ten wells of a standard 96-well plate with 100 µL low-sugar DMEM containing 10% FBS, and an equal amount of PBS solution was added to the remaining wells. After incubating overnight, the media in five out of ten wells were replaced with the diabetic microenvironment pretreatment medium, while that in the other five wells were replaced with the ordinary low-sugar DMEM containing 10% FBS. After incubating for 24 h, 20 µL methylthiazolyldiphenyl-tetrazolium bromide (MTT, Elabscience, Wuhan, China) solution (5 mg/mL, 0.5% MTT) was added to each well and incubated further for 4 h. The supernatant was aspirated, and the blue-purple precipitate at the bottom of the cell was saved. A total of 150 µL dimethyl sulfoxide (DMSO, Elabscience) was added to each well and the plate was shaken on a shaker at a low speed for approximately 10 min to fully dissolve the crystals. The absorbance of each well was measured at 490 nm using an enzyme-linked immunoassay (Thermo Scientific, CA, USA).

Cell proliferation was assessed by the cell counting kit 8 (CCK-8) assay. Cells were grown in 96-well culture plates at 8 × 10³ cells/well and treated with SFI at different drug concentrations (0, 0.04, 0.08 g/ml) for 24, 48 and 72 h. After treatment, the cells were incubated for 1 h at 37 °C with CCK-8 reagent (03.17002DA, EallBio). The absorbance of 96-well culture plates was tested at 450 nm by enzyme-linked immunoassay (Thermo, USA). All experiments were repeated at least three times.

The cultured H9C2 cells were seeded in 96-well culture plates, and after transfection and 1 μM ADR treatment, MTT solution (R&D Systems, Minneapolis, MN, USA) was added to each well. The cells continued to incubate for 4 hours in the incubator, then the supernatant of the 96-well plate was carefully discarded. 150 μL dimethyl sulfoxide (DMSO) (R&D Systems, Minneapolis, MN, USA) was added to each well and shaked for 10 minutes to fully melt the crystals. The activity of myocardial cells was detected, and the absorbance value of each well was measured by enzyme-linked immunoassay (Thermo Fisher Scientific, Waltham, MA, USA).