CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
For monitoring colocalization of mitochondria and autophagosomes, cells can be coincidently stained with MitoTracker Red CMXRos and the CYTO-ID.	Stain the cells with CYTO-ID for 1 h at 37°C in the dark	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.

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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Manufacturer protocol

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

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