CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	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.

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

Apoptosis was detected using Annexin V and PI kit (Abd Serotec), and tetramethylrhodamine, ethyl ester dye (TMRE, Sigma-Aldrich) according to the manufacturer's instruction. The apoptotic proportion was analyzed by Accuri™ C6 flow cytometer (BD Biosciences). To confirm whether ADI-PGE20-induced cell apoptosis is caspase dependent, the cells were treated with pan-caspase inhibitor Z-VAD-FMK (20 μM, Santa Cruz) in the presence of ADI-PEG20, stained with Annexin V/PI, and analyzed by flow cytometry FACS. For detection of autophagosomes, cyto-ID autophagy detection kit (Enzo Life Sciences) and Lyso Tracker Red (Life Technologies) were applied to this experiment.

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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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