Cell culture
MIN6 cells were maintained in DMEM with 15% heat inactivated FBS and 1% of antibiotic antimycotic mixture with 1X beta mercaptoethanol, in an incubator with temperature of 37°C and atmosphere of 5% CO2 and 95% humidity.
The cells were then trypsinised and seeded into a 12 well plate (1 × 105) and were pre-treated with naringin at the concentrations of 10 µM, 25 µM, 50 µM and 100 µM. After 12 h, the cells were treated with atorvastatin at the concentration of 200 µM. After an additional 16h, the cells were trypsinised for further downstream processing. The main stock of both compounds was prepared in DMSO (MP Biomedicals, Solon, Ohio, USA) from which working stock was diluted in DMEM (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA).
Mitochondrial membrane potential analysis using JC-1 dye
The trypsinised cells collected, pelleted, and washed with PBS. They were resuspended in 200 µL of the JC-1 solution (2 µM) (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA) at room temperature for 30 min. The cells were then washed following which 10,000 events were captured, on Accuri C6 flow cytometer (BD Biosciences Systems & Reagents Inc., San Jose, California, USA).
FITC Annexin V vs propidium iodide apoptosis detection analysis
For this purpose, the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences Systems & Reagents Inc., San Jose, California, USA) was used, and the proprietor recommended steps followed. Briefly, the cells were trypsinised washed with PBS, the cells were suspended in the FITC annexin V binding solution with the recommended dilution following which PI was added. After 15 min of staining, the sample was diluted to the run volume required and the sample was run using BD Accuri C6 flow cytometer with appropriate controls.