Hoechst reagent

Cells (2×10⁵) were plated in 24-well plates overnight and cultured in an incubator at 37°C prior to the experiment. MIN6 cells were washed in precooled 0.01 M PBS three times. Fresh 4% paraformaldehyde was used to fix the cells for 10 min at room temperature. Subsequently, the cells were permeabilized with 0.1% Triton-X 100 in 0.01 M PBS for 15 min at room temperature. Primary antibody appoptosin (cat. no. sc-515883; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and cleaved-caspase 3 (cat. no. 9661; Cell Signaling Technology, Inc., Danvers, MA, USA) were diluted to one hundred. Goat anti-rabbit IgG (Alexa Fluor^® 488; cat. no. A-11034; Thermo Fisher Scientific, Inc.) and Goat anti-mouse IgG (Alexa Fluor^® 594; cat. no. A-11020; Thermo Fisher Scientific, Inc.) were used as the secondary antibodies, which were diluted to one thousand. The detailed protocol of immunofluorescence has been described in our previous study (24 (link)). In brief, 10 µM JC-1 (Invitrogen; Thermo Fisher Scientific, Inc.) and 2 µg/ml Hoechst reagent (Guangzhou RiboBio Co., Ltd., Guangzhou, China) were added to MIN6 cells (90% fusion) for 10 min at 37°C at the same time. Cells were subsequently washed with warm DMEM. Finally, the stained cells were cultured in warm DMEM containing 10% FBS for live cell imaging (Confocal microscope FV1000; Olympus Corp., Tokyo, Japan).