CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
This kit uses a green fluorescent probe that reacts with autophagic vacuoles and is detectable by flow cytometry	10^7 fibroblast cells to stain with Cyto ID following digestion with trypsin. Autophagosomes can be photographed using fluorescence microscopy.	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
This kit uses a green fluorescent probe that reacts with autophagic vacuoles and is detectable by flow cytometry
Protocol tips
10^7 fibroblast cells to stain with Cyto ID following digestion with trypsin. Autophagosomes can be photographed using fluorescence microscopy.
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

Cells were grown to a number of 107, digested and washed twice with PBS (pH 7.4; HyClone; Thermo Fisher Scientific, Inc.), centrifuged at 175 × g for 5 min at 37°C, and resuspended in a PBS. The autophagosomes were marked and stained using the Cyto-ID Autophagy Detection kit (Enzo Biochem, Inc., New York, NY, USA) and photographed using a fluorescence microscope (Nikon Corporation, Tokyo, Japan). Fluorescence intensity was detected by flow cytometry (FC 500 MPL; Beckman Coulter, Inc., Brea, CA, USA).

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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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