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		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
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.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Autophagy assay cell type - HT29 using CYTO-ID® Autophagy detection kit from Enzo Life Sciences.

Manufacturer protocol

Download the product protocol from Enzo Life Sciences for CYTO-ID® Autophagy detection kit below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Autophagy assay cell type - HT29 using CYTO-ID® Autophagy detection kit from Enzo Life Sciences. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.