CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Use Rhodamine 123 along with CYTO-ID detection kit to see the co-localization of autophagosome and mitochondria.	Use Cyto-ID green dye (1 μl/4 ml assay buffer) for 30 minutes in dark at RT.	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.

Upstream tips
Use Rhodamine 123 along with CYTO-ID detection kit to see the co-localization of autophagosome and mitochondria.
Protocol tips
Use Cyto-ID green dye (1 μl/4 ml assay buffer) for 30 minutes in dark at RT.
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

Briefly, KB cells (1 × 106/35 mm dish) were incubated with FA-M-β-CyD (5 mM) for 2 h, in the presences and absence of pretreatment with 1 μM LY294002, an autophagic inhibitor, for 4 h, and then the cells were treated with Cyto-ID® Autophagy Detection Kit. A fluorescence microscope of Biozero BZ-8000 (KEYENCE) was used for cell observation.

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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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