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

HeLa pLuc/705 7 × 103 cells were seeded in a 96-well plate with glass bottom 24 h before treatment. Cells were incubated in serum-containing media with/without ligand for 1 h; then the fluorescent PF14/SCO(Alexa568) complexes (MR10, 100 nM SCO) were applied and incubated with cells for 24 h. Cells were then stained with Cyto-ID kit (Enzo Life Sciences) according to the manufacturer’s instruction. Imaging was performed using Leica DM/IRBE 2 epi-fluorescence microscope controlled by Micro-Manager19 with a 63 × 1.4 NA oil immersion objective. Images were analyzed using Fiji (ImageJ) software20. Quantification was performed by acquiring a stack with 0.3 µm slices through the cell. The stack was filtered with the 3D mean filter using 2 × 2 × 2 setting. Cells were then manually marked as a region of interest (ROI) and the signal from the ROIs with the highest average intensity used. Cell-free ROIs next to the measured cell was used for background subtraction.

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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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