CYTO-ID® Autophagy detection kit

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Chloroquine, a well-known reagent able to block autophagic flux and cause an accumulation of autophagosome in the cytoplasm, is used to treat Caco2 cells to use as positive control.

Upstream tips
Chloroquine, a well-known reagent able to block autophagic flux and cause an accumulation of autophagosome in the cytoplasm, is used to treat Caco2 cells to use as positive control.

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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