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

The cells were stained with Live-Dead to discriminate living cells. The following anti-human antibodies were used: anti-CD44 (1:1,000; Abcam, Cambridge, UK), anti-CD117 (non-activating AC126 clone, 1:10; Miltenyi-Biotec, Bergish Gladbach, Germany), anti-CD45 (1:10; Miltenyi-Biotec) and anti-Ki67 (1:10; BD Bioscience, Franklin Lakes, NJ, USA). All the cytofluorimetric analyses were performed using a FACS LSRII (BD); data were collected from at least 1 × 105 cells/sample and elaborated with FlowJo software (TreeStar, Ashland, OR, USA). For FACS sorting, antibody-labeled cells were separated with a MoFloAstrios Cell Sorter (Beckman Coulter, Brea, CA, USA); the purity of the sorted populations always exceeded 90%. To evaluate autophagic activity, EOC effusion cells were labeled with Cyto-ID Autophagy detection kit (Enzo Life Sciences).

For cell cycle analysis, cells were fixed with cold ethanol and then incubated for 1 h in a Dapi/RNAse solution. For cell viability analysis, cells were incubated for 15 min at 37 °C with AnnexinV/PI staining kit (Roche, Basel, Switzerland).

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Manufacturer protocol

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