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	After staining the cells with CYTO-ID detection reagent, treat the cells with 50nM Rapamycin which is an autophagy inducer (Treated cells can be used as positive control). - 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
This kit uses a green fluorescent probe that reacts with autophagic vacuoles and is detectable by flow cytometry
Protocol tips
After staining the cells with CYTO-ID detection reagent, treat the cells with 50nM Rapamycin which is an autophagy inducer (Treated cells can be used as positive control). - 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

Ramos and Daudi cells were treated with 40 ng/mL of Rituximab-MMAE for 24 h. Afterward, the cells were stained by Cyto-ID Autophagy Detection Kit (ENZO Life Science, Farmingdale, NY, USA) following the manufacturer’s instruction. Cells were then incubated with autophagy inducer, rapamycin (50 nM) for positive control and observed by inverted confocal microscope (Carl Zeiss LSM710, Carl Zeiss, Germany).

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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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