CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Stain the cells with CYTO-ID for 30 min at 37°C in the dark	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

Protocol tips
Stain the cells with CYTO-ID for 30 min at 37°C in the dark
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

The effect of Danu on cellular autophagy was further examined using confocal microscopy with the application of Cyto-ID® autophagy detection kit according to the manufacture’s instruction. The C13 and A2780cp cells were seeded into 8-well chamber slide at 30% confluence. After incubation overnight, the cells were treated with Danu at 0.01, 0.1, and 0.5 μM. After incubation for 24 h, 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 min 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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Manufacturer protocol

Download the product protocol from Enzo Life Sciences for CYTO-ID® Autophagy detection kit below.

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