CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
250 µL of the diluted Cyto-ID® green stain solution was added to each sample and mixed well instead of 100ul	Incubate for 30 minutes at 37°C 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
250 µL of the diluted Cyto-ID® green stain solution was added to each sample and mixed well instead of 100ul
Protocol tips
Incubate for 30 minutes at 37°C 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

The cells were treated with fresh medium alone, control vehicle alone (0.05% DMSO, v/v), or ALS at 0.1, 1, and 5 μM for 24 hours at 37°C. To investigate the mechanisms for ALS-induced autophagy, cells were pre-treated with 20 μM Z-VAD(OMe)-FMK, 10 μM WM, 0.1 μM bafilomycin A1, 30 μM chloroquine, 20 μM SB202190, or 25 nM FK866 for 1 hour, then treated with 5 μM ALS for a further 24 hours. Groups of cells treated with each of these compounds alone were also included. All inducers and inhibitors were dissolved in 0.05% DMSO. After ALS treatment, cells were re-suspended in 250 μL of phenol red-free culture medium (Invitrogen Inc.) containing 5% FBS, and 250 μL of the diluted Cyto-ID® green stain solution (Enzo Life Sciences Inc.) was added to each sample and mixed well. Cells were incubated for 30 minutes at 37°C in the dark, collected by centrifugation, washed with 1× assay buffer, and re-suspended in 250 μL fresh 1× assay buffer. Cells were finally analyzed using the fluorescein isothiocyanate channel of a flow cytometer that collected 10,000 events for analysis.

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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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