Low sugar dmem

The MIN6 cells were maintained in Dulbecco modified Eagle medium (DMEM; 1 g/L of glucose; Gibco, Grand Island, NY) supplemented with 15% FBS. Cells were incubated at 37°C in a 5% CO₂-humidified incubator. When cells reached 70% to 80% confluence, the culture medium was sucked out and changed to low sugar DMEM (1 g/L of glucose; Gibco), high sugar DMEM (4.5 g/L of glucose; Gibco), and 50 nM medium prepared from high sugar DMEM (containing 4.5 g/L of glucose; Gibco) + repaglinide (Sigma-Aldrich, St. Lousi, MO). After 15 minutes of stimulation, cells were collected for trichrome immunofluorescence staining (PCNT, INS, and F-actin), and the cell culture was collected to test for insulin concentration.

Four-week-old male SD rats were immersed in 75% ethanol for 10 min after cervical dislocation and then transferred to a clean bench. The lower femur and tibia of the rat were harvested, the bone ends of both sides were cut, and the medullary cavity was repeatedly washed with serum-free DMEM medium (Gibco). The obtained bone marrow cell suspension was transferred to a centrifuge tube and centrifuged at 1000 r/min for 5 min. The supernatant was discarded and the cells were resuspended in low sugar DMEM (Gibco) containing 10% fetal calf serum. The cells were seeded in 25 cm² medium and incubated in a 5% CO₂ incubator at 37°C. The culture solution was changed every 3 days. When the cells were fused to 80% to 90%, they were subcultured at 1:2. A 1×10⁹/L single cell suspension was prepared from the 4th generation cells. Flow cytometry (FACSCalibur, Becton-Dickinson, Franklin Lakes, USA) was used to detected the expression of CD29, CD34, CD44 and CD45 for MSCs identification.