Autophagy assay cell type - DM331

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 1 matching solution for this experiment

CYTO-ID® Autophagy detection kit

Enzo Life Sciences

Upstream tips	Protocol tips	Downstream tips
Kit contains specific dye that selectively stains autophagic compartments.	- 2 µL of CYTO-ID® Green Detection Reagent and 1 µL of Hoechst 33342 Nuclear Stain for every 1 mL of 1X Assay Buffer or complete cell growth medium	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
Kit contains specific dye that selectively stains autophagic compartments.
Protocol tips
- 2 µL of CYTO-ID® Green Detection Reagent and 1 µL of Hoechst 33342 Nuclear Stain for every 1 mL of 1X Assay Buffer or complete cell growth medium
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

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