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	Incubate for 30 minutes at 37°C in the dark	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
Incubate for 30 minutes at 37°C in the dark
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

MCF10A, MCF7, and MDA-MB-231 cells were treated with fresh medium alone, control vehicle alone (0.05% DMSO, v/v), or Danu (0.01, 0.1, and 0.5 μM) for 24 hours at 37°C. In separate experiments, MCF10A, MCF7, and MDA-MB-231 cells were treated with 0.5 μM Danu for 4, 8, 12, 24, 48, and 72 hours. To investigate the mechanisms for Danu-induced autophagy, cells were pretreated with 10 μM WM (a PI3K inhibitor and autophagy blocker), 10 μM bafilomycin A1 (an autophagy inhibitor), 10 μM SB202190 (a selective inhibitor of p38 MAPK), or 10 μM U0126 (a selective inhibitor of Erk1/2) for 30 minutes in MCF7 and MDA-MB-231 cells, then co-treated with 0.5 μM Danu for a further 24 hours. Groups of cells treated with each of these compounds alone were also included. All inhibitors were dissolved in DMSO at a final concentration of 0.05% (v/v). Cells were resuspended in 250 μL of phenol red-free culture medium (Thermo Fisher Scientific Inc.; No: 1294895) containing 5% FBS, and 250 μL of the diluted Cyto-ID® Green stain solution was added to each sample and mixed well. Cyto-ID® Green stain is a cationic amphiphilic tracer that selectively labels autophagic vacuoles in cells. Cells were incubated for 30 minutes at 37°C in the dark, collected by centrifugation, washed with 1× assay buffer, and resuspended in 500 μL fresh 1× assay buffer. Cells were subjected to flow cytometric analysis using the green (FL1) channel. The flow cytometer collected 10,000 events.

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Manufacturer protocol

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