CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
		- Incase of Low CYTO-ID Green dye staining in all treatments, increase the reagent concentration (500X dilution of the dye is recommended) and the incubation time. - Incase of High CYTO-ID Green staining, it could be that the cell culture medium was depleted of nutrients and so change media 4-8 hours before the experiment.

Downstream tips
- Incase of Low CYTO-ID Green dye staining in all treatments, increase the reagent concentration (500X dilution of the dye is recommended) and the incubation time. - Incase of High CYTO-ID Green staining, it could be that the cell culture medium was depleted of nutrients and so change media 4-8 hours before the experiment.

Publication protocol

To examine the effect of Danu on autophagy in C13 and A2780cp cells, cellular autophagy was detected using flow cytometry. Briefly, C13 and A2780cp cells were seeded in 60 mm Petri dishes for 24 h and the cells reached ~75% confluence, then treated with fresh medium alone, control vehicle alone (0.05% DMSO, v/v), or Danu (0.01, 0.1, and 0.5 µM) for 24 h. In separate experiments, C13 and A2780cp cells were treated with 0.5 μM Danu for 4, 8, 12, 24, 48, or 72 h. Following the Danu treatment, cells were resuspended in 250 μL of phenol red-free culture medium containing 5% FBS and 250 μL of the diluted Cyto-ID® Green stain solution was added to each sample and mixed well. Cells were incubated for 30 min at 37 °C in the dark and then collected by centrifugation at 250× g for 3 min. The cell pellet was washed with 1× assay buffer in the Cyto-ID® autophagy detection kit and resuspended in 500 μL fresh 1× assay buffer. Cells were analyzed using the green (FL1) channel of the flow cytometer.

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Manufacturer protocol

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