细胞实验
Cardiomyocytesisolation and culture[1]
Neonatal mouse cardiomyocytes were isolatedaccording to a previously described method, with little modification. In brief,hearts of neonatal mice were removed surgically, and the ventricles were enzymaticallydigested in 0.1% collagenase II for 25 min in a thermostat at 37OC.After collection, centrifugation, and resuspension, cells were pre-plated for 2hin DMEM supplemented with 15% FBS to reduce non-myocyte contamination and thenplated in culture dishes and incubated at 37OC in 5% CO2incubator. After 3–4 days, cells were incubated in a serum-free essentialmedium overnight before treatment with the indicated procedures. The cells werethen incubated in serum-containing medium, with a normal glucose concentration (5.5mMD-glucose) or HG concentration (33mM D-glucose) for 24h. ZLN005 treatment was appliedsimultaneously in specific groups (the concentration of ZLN005 was 4μM,unless noted otherwise).
Cellviability assay
Cell viability was measured by3-(4, 5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT).For the MTT assay, cells were plated on 96-wellplates at the density of 3X105cells/ml (1ml/well) prior to treatment.After treatment, MTT solution was added, and the cells were incubated for an additional4h. Then, the solution was aspirated, and DMSO was added to dissolve theformazan crystals. The final solution was transferred to a 96-well plate, and theabsorbance was measured at 570nm using a microplate absorbance spectrophotometer.Theresults were expressed as a percentage of control.
