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	Stain for 30 min at 37 C in dark.	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
Stain for 30 min at 37 C in dark.
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 on coverslips in a 24-well plate overnight. Cyto-ID® Autophagy Detection Kit (Enzo Life Sciences, Plymouth Meeting, PA, USA) was used to detect the expression of LC3 according to the manufacturer's protocol. In brief, cells were seeded on coverslips in 6-well plates. Following different treatments, cells were suspended with 1x Assay buffer and stained with Cyto-ID Green Detection Reagent and Hoechst 33342 Nuclear from light for 30 minutes at 37°C. The pictures were captured using confocal laser scanning microscope (Nikon, Tokyo, Japan) and processed using the software provided by the manufacturer.

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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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