CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream 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	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.

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

For experiments using MitoTempo as mitochondrial ROS scavenger, a final concentration of 25 nmol/L MitoTempo (Sigma Aldrich, St. Louis, MO, United States) was added to the cell culture 12 h prior to stimulation with HKC or E. coli. All cell nuclei were stained with DAPI (4’,6-Diamidino-2-Phenylindole, Dihydrochloride) for 15 min at room temperature. Autophagy experiments utilized the Cyto-ID® Autophagy Detection Kit (Enzo Life Sciences, Farmingdale, NY, United States) to detect autophagic vesicles and cells were processed according to manufacturer specifications. Briefly, cells were washed with assay buffer and incubated with a dual detection reagent containing Cyto-ID Green Detection Reagent and Hoechst stain for 30 min at 37 °C. After incubation, cells were washed with PBS and fixed with 4% paraformaldehyde. Cells were imaged immediately. Coverslips with cells for all experiments were mounted with Prolong® Gold anti fade mounting agent (Life Technologies Corp, Grand Island, NY, United States). Fluorescent images were taken at 60x (oil) magnification with a Zeiss Axiovert 200M and black and white AxioCam MRm (Zeiss) camera. All treatments were performed in duplicate and experiments were repeated a minimum of 3 times.

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Manufacturer protocol

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