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	Incubate cells with CYTO-ID dye (2μl of CYTO-ID in 1ml of 1X assay buffer) for 30 min	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
Incubate cells with CYTO-ID dye (2μl of CYTO-ID in 1ml of 1X assay buffer) for 30 min
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

HL‐1 cells transfected with Ehmt2 siRNA or negative control siRNA (as described above) were either treated with 5 μmol/L rapamycin for 18 hours or cultured in serum‐free medium (Hank's Balanced Salt Solution, Thermo Fisher) containing 10 μmol/L chloroquine for 4 hours to induce autophagy. Two separate experiments with at least 6 biological replicates in each treatment group were performed. Autophagic activity was probed using the CYTO‐ID Autophagy Detection Kit (Enzo Life Sciences, Farmingdale, NY) and measured with flow cytometry. For quantification of autophagy, cells were stained as per the manufacturer's instructions and run in a BD Accuri Flow Cytometer. The mean fluorescence intensity in FL‐1 was used as a measure of autophagic activity.

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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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