Autophagy/Cytotoxicity Dual Staining Kit

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This dual staining autophagy/cytoxicity cell-based assay facilitates the detection of both cells showing indicators of autophagy and cells showing indicators of cytotoxicity.

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This dual staining autophagy/cytoxicity cell-based assay facilitates the detection of both cells showing indicators of autophagy and cells showing indicators of cytotoxicity.

Publication protocol

The autophagy induction in SiO2 NPs treated MRC-5 cells was established using the Autophagy/Cytotoxicity Dual Staining Kit (Cayman Chemical, Ann Arbor, MI, USA). This dual staining autophagy/cytoxicity cell-based assay facilitates the detection of both cells showing indicators of autophagy and cells showing indicators of cytotoxicity. The monodansylcadaverine (MDC) is an autofluorescent compound which can incorporate into multilamellar bodies through an ion trapping mechanism and upon the interaction with membrane lipids, and thus can serve as a probe in the in vitro detection of autophagic vacuoles. Propidium iodide staining was performed as a marker of cell death. Being a well-known inducer of autophagy, tamoxifen was used as a positive control in this assay.

MRC-5 cells, cultured at a density of 5 × 104 cells per well in 96-well plates were treated with 62.5 µg/mL SiO2 NPs and different concentrations of tamoxifen (1–10 µM), for 24, 48, and 72 h. After the incubation period, the medium was removed, cells were washed with phosphate buffered saline (PBS) and then, a volume of 100 µL of appropriate diluted propidium iodide solution per well was added. After 10 min incubation at room temperature, the cells were washed with 100 µL wash buffer and treated for 10 min at 37 °C with 100 µL appropriate diluted solution MDC. Then the MDC solution was replaced with wash buffer and cells were immediately visualized by an Olympus IX71 fluorescence microscope (triple filter TRITC/FITC/DAPI, Olympus, Tokyo, Japan). The dead cells were revealed in red, while the autophagic vacuoles were shown in silver. The fluorescence was quantified using the image processing and analysis application, Image J.

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