CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Kit contains specific dye that selectively stains autophagic compartments.	- 2 µL of CYTO-ID® Green Detection Reagent and 1 µL of Hoechst 33342 Nuclear Stain for every 1 mL of 1X Assay Buffer or complete cell growth medium	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
Kit contains specific dye that selectively stains autophagic compartments.
Protocol tips
- 2 µL of CYTO-ID® Green Detection Reagent and 1 µL of Hoechst 33342 Nuclear Stain for every 1 mL of 1X Assay Buffer or complete cell growth medium
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

To detect MA flux, cells were incubated for 16 h at 37°C ± 20 μM CQ (Sigma-Aldrich, St. Louis, MO, United States) (Mizushima and Yoshimori, 2007; Mizushima et al., 2010; Klionsky et al., 2012). Western blotting was used to detect cellular LC3I and LC3II. Cellular LC3I and LC3II protein levels were normalized relative to actin protein levels to account for protein sample loading. MA flux was determined by subtracting the relative ratio of LC3II/actin in untreated cells from the relative ratio of LC3II/actin for CQ treated cells. To monitor MA in real time within live cells, melanoma cells were incubated 4 h at 37°C with media ± serum. Vesicles produced during MA in normal or starvation conditions were stained using CYTO-ID Autophagy detection kit (Enzo Life Sciences) and analyzed by flow cytometry (Guo et al., 2015).

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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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