CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Incubate cells at 30 min at 37 °C after adding the dye	Incase of Low dye staining in all treatments, increase the reagent concentration (500X dilution of the dye is recommended) and the incubation time. Incase of High 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
Incubate cells at 30 min at 37 °C after adding the dye
Downstream tips
Incase of Low dye staining in all treatments, increase the reagent concentration (500X dilution of the dye is recommended) and the incubation time. Incase of High 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

After treatment with siRNAs, HT1080-EGCP-LC3 cells were visualized using a fluorescence microscope (Carl Zeiss, Oberkochen, Germany, Axioscope). The cells were washed twice with PBS and fixed in paraformaldehyde (PFA) for 15 min. To observe lysosomes, the cells were incubated with 50–75 nM LysoTracker Red DND-99 (Thermo Fisher Scientific, L-7528) for 30 min at 37 °C. The number of autophagosome per cell and the size of autophagsome are measured by Operetta High-Content Imaging System and Harmony High-Content Imaging and Analysis Software (PerkinElmer, Waltham, MA, USA, HH12000000 and HH12000701), using Cyto-ID Autophagy Detection Kit (Enzo LifeScience, Farmingdale, NY, USA, ENZ-51031).

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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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