CYTO-ID® Autophagy detection kit

Autophagy assay cell type - HOS

Experiment
Autophagy assay cell type - HOS
Product
CYTO-ID® Autophagy detection kit from Enzo Life Sciences
Manufacturer
Enzo Life Sciences

Protocol tips

Upstream tips
For monitoring colocalization of mitochondria and autophagosomes, cells can be coincidently stained with MitoTracker Red CMXRos and the CYTO-ID.
Protocol tips
Stain the cells with CYTO-ID for 1 h 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

The mitochondrial network was analyzed as previously described (32) with minor modifications. Briefly, cells in FBS/DMEM (3×104/well) adherent on an 8-well chambered coverslip with a glass bottom (Imaging Chamber 8 CG; Zell-Kontakt GmbH, Nörten-Hardenberg, Germany) were treated with PSM (25 and 100%), 100 ng/ml TRAIL, 0.5 or 1 µM rapamycin and 5 mM 3-MA alone or in combination for 24 h in a humidified atmosphere containing 95% air/5% CO2 at 37°C. Following media removal by aspiration, the cells were washed with fresh FBS/DMEM and stained with 20 nM MitoTracker Red CMXRos for 1 h at 37°C in the dark. The nuclei were counterstained with Hoechst 33342. The cells were then washed with and immersed in FluoroBrite™ DMEM (Thermo Fisher Scientific, Inc.). Images were obtained using a BZ X-700 Fluorescence Microscope (Keyence Corporation, Osaka, Japan) equipped with a 100X, 1.40 n.a. UPLSAPO super-apochromat, coverslip-corrected oil objective (Olympus Corporation, Tokyo, Japan). Images were analyzed using BZ-H3A application software (Keyence Corporation) and National Institutes of Health (NIH) ImageJ software (bundled with 64-bit Java 1.8.0_112; NIH, Bethesda, MD, USA). The formation of autophagosomes was analyzed using the CYTO-ID® Autophagy Detection kit (Enzo Life Sciences, Inc.) according to the manufacturer's protocol. Briefly, cells were stained with CYTO-ID for 1 h at 37°C in the dark and treated with the aforementioned agents. For monitoring colocalization of mitochondria and autophagosomes, cells were coincidently stained with MitoTracker Red CMXRos and the CYTO-ID. Images were obtained using EVOS FL Cell Imaging system (Thermo Fisher Scientific, Inc.) equipped with a 40X, 0.60 n.a. LUCPLFLN objective (Olympus Corporation) as previously described (33).

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Papers

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Manufacturer protocol

Download the product protocol from Enzo Life Sciences for CYTO-ID® Autophagy detection kit below.

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