CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
This dye specifically accumulates in the autophagosomes allowing the evaluation of the extent of autophagy as number of green fluorescent spots into each single cell.	Use Cyto-ID green dye (1 μl/4 ml assay buffer) for 30 minutes in dark at RT	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 dye specifically accumulates in the autophagosomes allowing the evaluation of the extent of autophagy as number of green fluorescent spots into each single cell.
Protocol tips
Use Cyto-ID green dye (1 μl/4 ml assay buffer) for 30 minutes in dark at RT
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 detected in MCL cells infected or not with LC3-GFP retroviral expression vector. 106 cells were labeled using the Cyto-ID® Autophagy Detection Kit (Enzo Life Sciences) according to manufacturer's instructions. Cells undergoing autophagy showed green fluorescent countable punctuate structures. Similarly, in cells infected with LC3-GFP retroviral expression vector, the autophagy extent is measurable through the counting of LC3-GFP puncta per cell. 50×103 cells/sample were acquired with the ImageStreamX instrument (Amnis Corporation, Seattle, WA) using the INSPIRE software and, using a specific feature of the IDEAS analysis software, the number of fluorescent spots per cell was evaluated. The “H variance mean” algorithm of the IDEAS software allowed to evaluate the distribution and the texture of fluorescence into the cells also in the presence of high background.

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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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