CYTO-ID® Autophagy detection kit

Protocol tips

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	Add 250 μL of diluted Cyto-ID Green stain solution to samples and incubate for 30min at 37°C in the dark

Protocol tips
Add 250 μL of diluted Cyto-ID Green stain solution to samples and incubate for 30min at 37°C in the dark

Publication protocol

Determination of cellular autophagy
To determine the effect of danusertib on autophagy in AGS and NCI-N78 cells, the intracellular autophagy level was examined using flow cytometry as previous described.21 Briefly, the cells were treated with fresh medium alone, control vehicle alone (0.05% dimethyl sulfoxide, v/v), or danusertib at 0.01, 0.1, and 0.5 μM for 24 hours. After 24 hours of incubation, the cells were trypsinized and collected with 1× assay buffer containing 5% fetal bovine serum. The cells were resuspended in 250 μL of phenol red-free culture medium (Invitrogen Inc) containing 5% fetal bovine serum, and 250 μL of diluted Cyto-ID Green stain solution was added to each sample and mixed well. The cells were incubated for 30 minutes at 37°C in the dark. Following incubation, the cells were collected by centrifugation at 250× g for 3 minutes and washed with 1× assay buffer. Subsequently, the cells were resuspended in 500 μL of fresh 1× assay buffer containing 5% fetal bovine serum and subject to flow cytometric analysis within one hour of adding they assay buffer. Cells were analyzed using the green (FL1) channel of a flow cytometer.

Confocal fluorescence microscopy
Confocal microscopic analysis was performed to further examine the cellular autophagy level and the mechanisms of danusertib-induced autophagy in AGS and NCI-N78 cells using a Cyto-ID autophagy detection kit. Briefly, AGS and NCI-N78 cells were seeded into an 8-well chamber slide at 30% confluence. The cells were treated with danusertib at 0.01, 0.1, and 0.5 μM for 24 hours. In separate experiments, to investigate the mechanisms for danusertib-induced autophagy, cells were pretreated with 10 μM WM (a PI3K inhibitor and autophagy blocker) and 10 μM SB202190 (a selective inhibitor of p38 MAPK used as an autophagy inducer), and then cotreated with 0.5 μM danusertib for a further 24 hours. After incubation for 24 hours, the cells reached ~60% of confluence and were washed with 1× assay buffer, following by incubation with 100 μL of microscopy dual detection reagent for 30 minutes at 37°C in the dark. After incubation, the cells were washed with 1× assay buffer to remove the detection reagent, and then examined using a TCS SP2 laser scanning confocal microscope (Leica, Wetzlar, Germany) using a standard fluorescein isothiocyanate filter set for imaging the autophagic signal at wavelengths of 405/488 nm

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