Monodansylcadaverine mdc kit

To visualize the autophagic process in S. sclerotiorum, monodansylcadaverine (MDC) was used as a fluorescent pigment to detect specific markers of autophagosome formation. The autophagosomes were observed in the mycelia of WT and ΔssAtg1 cultured in liquid CM at 25 °C for 24 h, then transferred to liquid MM-N for 6 h, the monodansylcadaverine (MDC) kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) was used to stain autophagosome, and then viewed under epi-fluorescence microscopy.

Autophagic vesicles in response to different treatments were analyzed by using the monodansylcadaverine (MDC) kit (Solarbio, Beijing, China). After treatments, the cardiomyocytes were stained with MDC following the manufacturer's protocol. Briefly, 300 μL of the staining solution was added to the cells and incubated for 45 minutes at room temperature. Then, the cells were washed twice with washing buffer and examined by fluorescence microscopy with a 355 nm excitation wavelength and a 512 nm emission filter. The intensity of immunofluorescence staining was quantified using ImageJ software (NIH, Maryland, USA).

Cells after treatment were collected and washed in PBS 3 times. They were resuspended in loading buffer (Sangon Biotech, Shanghai, China) to 2 × 105 to 5 × 105 cells/mL. For apoptosis and autophagy analysis, Annexin V-FITC/PI kit (Sangon Biotech) and monodansylcadaverine (MDC) kit (Beijing Solarbio Science & Technology Co. Ltd., Beijing, China) were utilized, respectively. BD FACSCanto™ II system (BD Biosciences, San Jose, CA, USA) was utilized to detect cell surface markers. All the results were analyzed by the FlowJo software.