CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- Add 250 μL of diluted Cyto-ID Green stain solution to samples and incubate for 30min at 37°C in the dark

Protocol tips
- Add 250 μL of diluted Cyto-ID Green stain solution to samples and incubate for 30min at 37°C in the dark

Publication protocol

Confocal microscopic analysis was performed to further examine the cellular autophagy level and the mechanisms of danusertib-induced autophagy in AGS and NCI-N78 cells using a Cyto-ID autophagy detection kit. Briefly, AGS and NCI-N78 cells were seeded into an 8-well chamber slide at 30% confluence. The cells were treated with danusertib at 0.01, 0.1, and 0.5 μM for 24 hours. In separate experiments, to investigate the mechanisms for danusertib-induced autophagy, cells were pretreated with 10 μM WM (a PI3K inhibitor and autophagy blocker) and 10 μM SB202190 (a selective inhibitor of p38 MAPK used as an autophagy inducer), and then cotreated with 0.5 μM danusertib for a further 24 hours. After incubation for 24 hours, the cells reached ~60% of confluence and were washed with 1× assay buffer, following by incubation with 100 μL of microscopy dual detection reagent for 30 minutes at 37°C in the dark. After incubation, the cells were washed with 1× assay buffer to remove the detection reagent, and then examined using a TCS SP2 laser scanning confocal microscope (Leica, Wetzlar, Germany) using a standard fluorescein isothiocyanate filter set for imaging the autophagic signal at wavelengths of 405/488 nm.

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