Evaluating C2C12 Cell Viability under DPHC

Previous studies have shown that C2C12 cells represent a model to study insulin resistance in diabetes and improvement in muscle function [21 (link), 22 (link)]. C2C12 skeletal muscle cells (American Type Culture Collection) were maintained in DMEM with 10% FBS and antibiotics, at 37°C in a humidified incubator of 5% CO₂. When the cells reached 80% confluence, C2C12 cells were differentiated into skeletal myotubes in DMEM-low glucose with 2% horse serum for 5 days. Viability levels of C2C12 cells were determined by the ability of mitochondria to convert MTT to an insoluble formazan product. Cells were maintained in 96-well plates at a density of 3 × 10⁴ cells/well and subsequently subjected to different concentrations of DPHC (0.1, 2, 10, and 30 μM) for 24 h. Cells were pretreated with MTT solution (2 mg/mL) for 3 h. Subsequently, cell density was determined by measuring optical density (OD) at 540 nm using a microplate reader (Gen5 version 2.05, BioTek, Winooski, Vermont, USA).