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	Resuspend 5 × 10*5 cells in 1× assay buffer and incubate with the Cyto‐ID dye at 37 ºC for 30 minutes in the dark, followed by flow‐cytometric analysis. For Fluorescence microscopy, grow the cells on coverslips after respective treatment incubate them with the dual detection reagent (Cyto‐ID dye and Hoechst 3342 nuclear stain) at 37 ºC for 30 minutes in the 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
Resuspend 5 × 10*5 cells in 1× assay buffer and incubate with the Cyto‐ID dye at 37 ºC for 30 minutes in the dark, followed by flow‐cytometric analysis. For Fluorescence microscopy, grow the cells on coverslips after respective treatment incubate them with the dual detection reagent (Cyto‐ID dye and Hoechst 3342 nuclear stain) at 37 ºC for 30 minutes in the 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

Autophagy was determined either by quantifying the LC3‐II/actin ratio following western blotting (Supporting Information) or using the Cyto‐ID Autophagy detection kit (Enzo Life Sciences, Farmingdale, NY) according to the manufacturer's instructions. Briefly, HepG2 cells were treated with wtAAV2 or drugs for specific time periods. After trypsinization and washing, 5 × 105 cells were resuspended in 1 × assay buffer and incubated with the Cyto‐ID dye at 37 ºC for 30 minutes in the dark, followed by flow‐cytometric analysis. For fluorescence microscopy, cells were grown on coverslips, treated with wtAAV2 or drugs for specific time periods, washed, and incubated with the dual detection reagent (Cyto‐ID dye and Hoechst 3342 nuclear stain) at 37 ºC for 30 minutes in the dark. After washing, cells were fixed in 4% paraformaldehyde and analyzed by fluorescence microscopy using the Olympus IX81 equipped with Cell Imaging software (Tokyo, Japan). Images were analyzed by CellProfiler 2.4.0rc1 (rev29342ff). Mean fluorescence intensities of Cyto‐ID per cell were quantified using at least 100 cells from each individual experiment. The autophagy activity factor was calculated in both flow‐cytometric and fluorescence microscopic analyses as described in the Supporting Information.

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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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