CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Use 2 μM ATRA treated Saos cells as positive control. ATRA is known to modulate autophagy in this cell line.		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
Use 2 μM ATRA treated Saos cells as positive control. ATRA is known to modulate autophagy in this cell line.
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 monitored using the Cyto-ID Autophagy Detection Kit (ENZO Life Science, Milan, Italy) as described [24, 47, 48]. Caco-2 and SAOs cells were incubated for 168 h with CEN (400 and 200 μg/ml (w/v), resp.) and with 20 μM chloroquine or 2 μM ATRA as positive controls. In particular, we estimated the impaired autophagy flux in SAOs cells by monitoring the accumulation of autophagic compartments induced by chloroquine, a lysosome inhibitor that impedes the fusion of autophagosomes and lysosomes and/or the activity of autolysosomes, adding it the last 4 h of incubation together with different treatments as described [49, 50]. After incubation, cells were washed and incubated with the autophagy detection marker (Cyto-ID) and the nuclear dye (Hoechst 33342). Subsequently, cells were rinsed with assay buffer and photographed using a fluorescence microscope (Zeiss Axiovert 200) and a confocal microscopy (Leica SP8; Leica Microsystems, Milan, Italy). Finally, autophagosomes were quantified by normalizing green (Cyto-ID) and blue (Hoechst) fluorescences using a microplate fluorescence reader (Synergy HT BioTek).

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Manufacturer protocol

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