Autophagy assay cell type - A2058

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 2 matching solutions for this experiment

Upstream tips
- Cells should be healthy and not overcrowded as results of the experiments will depend significantly on the cells’ condition.
Downstream tips
- Incase of Low CYTO-ID Green
dye staining in all treatments, increase the Increase the reagent concentration (500X dilution of the dye is
recommended) and increase 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.
Protocol tips
- Make 1:500 dilution of primary antibody
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