CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
500 nM rapamycin and 10 μM chloroquine treated cells can be used as positive control		Incase of Low CYTO-ID 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 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
500 nM rapamycin and 10 μM chloroquine treated cells can be used as positive control
Downstream tips
Incase of Low CYTO-ID 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 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

N2a cells (2×104) were seeded onto glass coverslips in 24-well plates and incubated overnight. The cells were treated with various concentrations of malathion (0, 0.25, 0.5, or 1 mM) for 8 h. Autophagic induction was detected using the Cyto-ID autophagy detection kit. Along with malathion, cells were also treated with a mixture of 500 nM rapamycin and 10 μM chloroquine, included in the kit, as positive controls. Experiments were carried out according to the manufacturer’s instructions. Fluorescent images were captured with a confocal laser scanning microscope (Nikon A1+) with excitation and emission wavelengths of 488 and 530 nm, respectively. The induction of autophagy was assessed by the intensity of green fluorescence in the cytosol.

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Manufacturer protocol

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