Ultra 897 ccd

To evaluate ROS generation, we used a modified method as described previously [8] . Briefly, cells were cultured in glass-bottom dishes and loaded with 2.5 μmol/L of H2DCFDA (Thermo fisher scientific, Waltham, MA, USA) for 20 min. The dishes were then mounted on a Carl Zeiss Axio Observer Z1 microscope with a Sutter Lambda DG-4 monochromator and filter sets. Cells were illuminated at a very low light intensity of 488 nm, and pictures were taken at 10 frames/s using a 40× oil immersion objective at 510 nm with an iXon Ultra 897 CCD. The rates of change of the DCF fluorescence signal within a single cell were calculated to report the rate of ROS generation. This method can avoid the difference in DCF loading and resting signal between cells.

Autophagic flux was measured as described previously [20] . Briefly H9c2 cells were plated on the coverslips and incubated with an mRFP-GFP-LC3 lentivirus (MOI 15, Hanbio. Co. LTD, Shanghai, China). After 36 h of infection, confocal images were obtained at 561 and 488 nm using a 63× oil immersion objective (iXon Ultra 897 CCD). The puncta of each cell were counted, and 10 pictures were taken for each sample. The GFP signal is sensitive to the acidic conditions of the lysosome lumen, whereas mRFP is more stable. Therefore, colocalization of both GFP and mRFP fluorescence indicates a compartment that has not fused with a lysosome. In contrast, an mRFP signal without GFP indicates a compartment fused with a lysosome. So, autophagy flux was then measured by confocal counting of GFP+/mRFP+(yellow) and GFP-/mRFP+(red) puncta [7] .