Dansylcadaverine

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- Use 0.05 mM MDC after 24h of culturing to and incubate for 15min

Protocol tips
- Use 0.05 mM MDC after 24h of culturing to and incubate for 15min

Publication protocol

Monodansylcadaverine (Sigma-Aldrich, 30432) is a marker of autophagic vacuoles.33 The C6 cells were seeded onto 6-well plates with a slide. The cells were incubated on the glass slides after the addition of different processing factors. After 24 h of incubation, 0.05 mM MDC was added to the culture medium and incubated for 15 min to stain autophagic vacuoles. Then, the glioma cells were rinsed 3 times with PBS and examined using a fluorescence microscope (Olympus IX51, Tokyo, Japan).

The Lipofectamine™ 2000 transfection reagent (Invitrogen, 11668019) was used to transiently transfect mCHERRY-EGFP-Lc3b (Addgene, 22418; deposited by Jayanta Debnath [University of California at San Francisco, USA]) into C6 cells. After 6-well plates were incubated at 37°C and 5% CO2 for 6 h, the medium was replaced with culture medium supplemented with fetal bovine serum. After different processing factors were added, the cells were imaged using a fluorescence microscope to calculate the number of cells that had been transfected.

Approximately 3000 C6 cells per well were seeded onto 96-well plates. After approximately 24 h, the cell density reached the 70% to 80% that was required for the experiments. A nanomaterial treatment group, a negative control group, and a positive control group were established; the control group was treated with a volume of PBS buffer equal to the volume of material that was added in the other groups. Half an hour earlier than the specified time, Rosup (Beyotime, S0033) was added to positive control wells and incubated for half an hour to produce ROS; dichlorofluorescin (Beyotime, S0033) was then added to all wells, including the positive and negative controls, until a predetermined time. Diacetate (Beyotime, S0033) was added to make a final concentration of 50 nM. The cells were then incubated for 20 min. Serum-free medium or PBS buffer was used to wash the cells twice, and the cells were then imaged using an inverted fluorescence microscope.

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