Autophagy assay cell type - LN229

As autophagy is a multi-step process which includes not just the formation of autophagosomes, but most importantly, flux through the entire system, including the degradation upon fusion with lysosomes, which makes it quite challenging for detection. There are several methods for detection in mammalian cells, including immunoblotting analysis of LC3 and p62 and detection of autophagosome formation/maturation by fluorescence microscopy, Currently, there is no single “gold standard” for determining the autophagic activity that is applicable in every experimental context, hence it is recommended to go for the combined use of multiple methods to accurately assess the autophagic activity in any given biological setting.

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Found 3 matching solutions for this experiment

CYTO-ID® Autophagy detection kit

Enzo Life Sciences

Upstream tips	Protocol tips	Downstream tips
This kit uses a green fluorescent probe that reacts with autophagic vacuoles and is detectable by flow cytometry	Incubate cells with CYTO-ID dye (2μl of CYTO-ID in 1ml of 1X assay buffer) for 30 min	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
This kit uses a green fluorescent probe that reacts with autophagic vacuoles and is detectable by flow cytometry
Protocol tips
Incubate cells with CYTO-ID dye (2μl of CYTO-ID in 1ml of 1X assay buffer) for 30 min
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

Manufacturer protocol Publication protocol 1 Relevant paper

Acridine Orange hemi(zinc chloride) salt

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
Intercalates between DNA/RNA and used to analyze mitochondria and Lysosomal content by flow cytometry.	For acidic vesicular organelle (AVO) staining, incubate cells at 37°C for 15 minutes with acridine orange (1 μg/mL) containing medium. Analyze the cells under FACS.

Upstream tips
Intercalates between DNA/RNA and used to analyze mitochondria and Lysosomal content by flow cytometry.
Protocol tips
For acidic vesicular organelle (AVO) staining, incubate cells at 37°C for 15 minutes with acridine orange (1 μg/mL) containing medium. Analyze the cells under FACS.

Manufacturer protocol Publication protocol 1 Relevant paper

Autophagy Inhibitor, 3-MA

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
It is an autophagosome inhibitor.Dissolve it in PBS.	Use 30mg/kg/d for in vivo treatment in mice and 2nM for in vitro application in cell line

Upstream tips
It is an autophagosome inhibitor.Dissolve it in PBS.
Protocol tips
Use 30mg/kg/d for in vivo treatment in mice and 2nM for in vitro application in cell line

Manufacturer protocol Publication protocol 1 Relevant paper

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