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	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
This kit uses a green fluorescent probe that reacts with autophagic vacuoles and is detectable by flow cytometry
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

HCT116, HT29, and SW620 cells were treated with either 0-114.6 μM euglenophycin, 0.5 μM rapamycin, or 100 μM chloroquine for 48-72 hr. Autophagy was detected using CYTO-ID autophagy detection kit (Enzo Life Sciences; Farmingdale, NY) following the manufacturer’s recommended protocol. All samples were analyzed in the FITC-channel using a BD LSR II flow cytometer (BD Biosciences, San Jose, CA). Data analysis with doublet discrimination was done using FlowJo version 7.6.5 software.

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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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