CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Stain the cells with CYTO-ID for 30min at RT in the dark	Incase of Low CYTO-ID 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 staining, it could be that the cell culture medium was depleted of nutrients and so change media 4-8 hours before the experiment.

Protocol tips
Stain the cells with CYTO-ID for 30min at RT in the dark
Downstream tips
Incase of Low CYTO-ID 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 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

Quantification of cellular autophagy
For autophagy detection, SCC25 cells were seeded in six-well plates and allowed to attach overnight. The cells were then treated with vehicle (0.05% DMSO, v/v) or PLB at concentrations of 0.1, 1, and 5 μM. In separate experiments, SCC25 cells were treated with 5 μM PLB for 4, 8, 24, 48, and 72 hours. Each live cell sample was trypsinized, washed by resuspending the cell pellet in 1× assay buffer (No ENZ-51031-K200; Enzo Life Sciences Inc.) and collected by centrifugation. Cells were resuspended in 250 μL of phenol red-free culture medium (No 1294895; Invitrogen, Carlsbad, CA, USA) containing 5% FBS, and 250 μL of the diluted Cyto-ID® Green stain solution (No ENZ-51031-K200; Enzo Life Sciences Inc.) was added to each sample and mixed well. Cells were incubated for 30 minutes at room temperature in the dark, collected by centrifugation, washed with 1× assay buffer, and resuspended in 500 μL fresh 1× assay buffer. Cells were analyzed using the green (FL1) channel of a flow cytometer (BD™ LSR II Analyzer).

Confocal fluorescence microscopy
The cellular autophagy level was examined using the Cyto-ID® autophagy detection kit according to the manufacturer’s instructions. Briefly, SCC25 cells were seeded in an eight-well chamber slide at 30% confluence. The cells were treated with PLB at 0.1, 1, and 5 μM for 24 hours. In separate experiments, SCC25 cells were treated with 5 μM PLB for 4, 8, 24, 48, and 72 hours. When the cells reached ~60% confluence, they were washed by 1× assay buffer, following by incubation with 100 μL of microscopy dual detection reagent for 30 minutes at 37°C in the dark. After incubation, the cells were washed with 1× assay buffer to remove detection reagent and then the cells were examined using a Leica TCS SP2 laser scanning confocal microscopy (Leica Microsystems, Wetzlar, Germany) using a standard fluorescein isothiocyanate (FITC) filter set for imaging the autophagic signal at wavelengths of 405/488 nm.

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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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