CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- Stain the cells for 1 hour at 37 C in dark.

Protocol tips
- Stain the cells for 1 hour at 37 C in dark.

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).

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Autophagy assay cell type - 143B using CYTO-ID® Autophagy detection kit from Enzo Life Sciences.

Manufacturer protocol

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

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Autophagy assay cell type - 143B using CYTO-ID® Autophagy detection kit from Enzo Life Sciences. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.