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	Following treatment, wash the cells in phosphate buffered saline, and resuspend in 1000× dilution of the detection reagent. Incubate for 30 min at 37 °C, wash the cells and analyse by flow cytometry.	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
Following treatment, wash the cells in phosphate buffered saline, and resuspend in 1000× dilution of the detection reagent. Incubate for 30 min at 37 °C, wash the cells and analyse by flow cytometry.
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 was evaluated using the FlowCellect™ Autophagy LC3 Antibody-based Assay kit (Merck Millipore) according to manufacturer’s instructions46. In brief, discrimination between cytosolic- and autophagosome associated LC3 is achieved by monitoring the translocation of LC3 using flow cytometry. Because autophagy is a constitutive cellular degradation process, pre-treatment with lysosomal inhibitor is required for 30 min prior to treatment of the 72-h-cultured sample with anti-LC3 FITC to prevent lysosomal degradation of LC3. Quantification of fluorescence can then be performed using anti-LC3 FITC. Cytosolic and autophagosomic populations are differentiated by washing cells to remove cytosolic LC3-I and retaining only membrane-bound LC3-II prior to staining.

Cellular autophagic flux (turnover) was also monitored using a Cyto-ID autophagy detection kit (Enzo Life Sciences, Farmingdale, NY, USA) according to the manufacturer’s instructions28,47. In brief, the 488-nm excitable Cyto-ID green autophagy detection reagent supplied with the kit becomes brightly fluorescent in vesicles produced during autophagy serving as a convenient tool to detect autophagy at a cellular level. Following treatment, the cells were washed in phosphate buffered saline, and resuspended in 1000× dilution of the detection reagent. After 30 min of incubation at 37 °C, the cells were washed and analysed by flow cytometry.

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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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