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	Incubate cells with CYTO-ID dye (2μl of CYTO-ID in 1ml of 1X assay buffer) for 30 min	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
Protocol tips
Incubate cells with CYTO-ID dye (2μl of CYTO-ID in 1ml of 1X assay buffer) for 30 min
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 hFOB 1.19 cells (1 × 104) were seeded on a Lab‐Tek™ II Chamber Slide (Nunc; Thermo Fisher Scientific, Rochester, NY) for 24 h. The next day, the cells were treated with galangin (5 µM), autophagy inhibitor 3‐MA (500 µM), and autophagy inducer rapamycin (50 nM) for 24 h. CYTO‐ID® Green autophagy dye was used to detect autophagy in live cells.26 The autophagosomes and autolysosomes were stained using a CYTO‐ID® Autophagy Detection Kit (Enzo Life Sciences, Farmingdale, NY) and analyzed using confocal microscopy (Carl Zeiss, Germany). Staining was conducted following manufacturer’ protocol

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Manufacturer protocol

Download the product protocol from Enzo Life Sciences for CYTO-ID® Autophagy detection kit below.

Download manufacturer protocol

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