CYTO-ID® Autophagy detection kit

Protocol tips

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	Cyto-ID dye dilution 1:4000 , staining for 30 min at 37degree in dark. No washing of cells needed for acquisition in FlowCytometry.

Protocol tips
Cyto-ID dye dilution 1:4000 , staining for 30 min at 37degree in dark. No washing of cells needed for acquisition in FlowCytometry.

Publication protocol

The Cyto-ID Autophagy Detection Kit (Enzo Life Sciences, Farmingdale, NY, USA) was used to detect AP 5. Briefly, CAMs (1 × 105/ml) were collected and centrifuged (400 × g, 5 min.) at the end of the treatment. Then, CAMs were incubated with 0.5 ml of freshly diluted Cyto-ID Green Detection Reagent (1:4000) for 30 min. at 37°C in the dark. Without washing, stained CAMs were run in the green (FL1) channel with a Guava Easycyte Mini Flow Cytometry System (Guava Technologies, Hayward, CA, USA) and analysed with Guava acquisition and analysis software (Guava Technologies). The enhancement of Cyto-ID Green dye signal indicates an increase in AP.

In addition, acridine orange (Sigma-Aldrich, St. Louis, MO, USA) was used to detect APLs. CAMs (1 × 106/ml) were stained with acridine orange (1:5000) for 17 min. After washes, CAMs were harvested in phenol red-free growth medium. Green (510–530 nm) and red (>650 nm) fluorescence emission from 104 cells illuminated with blue (488 nm) excitation light was measured with Flow Cytometry System and analysed with Guava acquisition and analysis software. The mean red/green fluorescence ratio was calculated to indicate the change of intracellular APLs.

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Manufacturer protocol

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