2.4　Immunofluorescence staining

Immunofluorescence staining was used to assess autophagosome formation.MIN-6 cells were cultured in 12 well plates which placed round glass coverslips in advance，treated with different conditions of the basic medium，basic medium with 33.3mmol/L glucose，basic medium with 1μL/mL XKP mixture；basic medium with 33.3mmol/L glucose and 1μL/mL XKP mixture；or basic medium with 33.3mmol/L glucose，1μL/mL XKP mixture and 2mmol/L 3-MA.After 36 h of incubation at 37℃，the medium was discarded.The cells were washed with PBS three times，fixed with 4% paraformaldehyde for 10min，and then washed with PBS three times.After blocking with blocking liquid(PBS plus 10% FBS)for 30min，cells were incubated at 37℃for 1 h with a primary antibody，anti-LC3(microtubule-associated protein 1 light chain 3)polyclonal antibody(MBL，PM036)，and then washed three times with blocking liquid.Cells were subsequently incubated 37℃for 1 h with a secondary antibody，anti-rabbit IgG Alexa Fluor 488 conjugate(CST，4412)，and then washed three times with PBS.Imaging was performed with a confocal laser scanning microscopy(Zeiss LSM 710).(https://www.daowen.com)