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

Autophagy levels were measured using two distinct assays. The first method involved using a lysotropic dye, acridine orange, which accumulates in acidic organelles in a pH-dependent manner, becomes protonated and trapped, and emits a bright red fluorescence. The red fluorescence was then detected by fluorescence-activated cell sorting (Coulter, FL2 channel). Bafilomycin A1 (Sigma Chemical Co.) was dissolved in DMSO and added to the cells 30 min before the addition of acridine orange. The second method employed the use of the CYTO-ID® Autophagy detection kit (Enzo Life Sciences, Inc) according to the manufacturer's protocol. Cells were then analyzed by fluorescence microplate reader.

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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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