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		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
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

Cyto-ID labeling and Flow cytometry: HEK-293 cells were harvested in Accutase solution (Merck Millipore, SF006), rinsed in DPBS and incubated for 30 min at room temperature in DMEM without phenol red, containing 5% fetal bovine serum and the fluorescent Cyto-ID probe (Cyto-ID® Autophagy detection kit; Enzo Life Sciences, ENZ-51031K200). After incubation, cells were washed and analyzed on a FACScalibur flow cytometer (BD Biosciences) operated with the CellQuest software. hrGFP-WIPI1 and EGFP-LC3B redistribution: cells expressing either hrGFP-WIPI1 or EGFP-LC3B were fixed in paraformaldehyde (4 %, 10 min) and imaged by confocal microscopy (Leica TCS SP2 confocal laser scanning microscope). The number of hrGFP-WIPI1 and EGFP-LC3B dots was assessed using the Imaris software. SQSTM1/p62 accumulation: autophagic flux was assessed by immunocytochemical analysis of endogenous SQSTM1 levels as described above. The integrated densities obtained from the green channel in 20 different images were normalized to the number of nuclei (DAPI stained) and represent levels of the SQSTM1 protein.

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Manufacturer protocol

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