CYTO-ID® Autophagy detection kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Culture cells for 24 hours after treating with CoCl2 (300μM) as well as Lutein (20μM).	- Stain cells with Cyto-ID® Green Detection Reagent for 45 minutes at 37°C

Upstream tips
- Culture cells for 24 hours after treating with CoCl2 (300μM) as well as Lutein (20μM).
Protocol tips
- Stain cells with Cyto-ID® Green Detection Reagent for 45 minutes at 37°C

Publication protocol

Assessment of autophagic activity in rMC-1 cells was performed using Cyto-ID® Autophagy Detection kit (Enzo, Life Science). In brief, cells were grown on a chamber slide and treated with CoCl2 (300μM) as well as Lutein (20μM) for 24 hours. Cells co-treated with 0.5μM rapamycin and 120μM chloroquine for 16 hours were used as the positive control to induce autophagy and to identify the autophagic vesicles in rMC-1 cells. After washes by assay buffer, cells were incubated with Cyto-ID® Green Detection Reagent for 45 minutes at 37°C. Hoechst 33342 was used as a nuclei counterstain. Eight non-overlapping fields were photographed in each group using a light microscope (Eclipse 80i Nikon, Tokyo, Japan) equipped with a digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI) and analyzed with Image J software (National Institute of Mental Health, Bethesda, MD). At least three independent experiments were performed.

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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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