Autophagy assay cell type - N2a

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
500 nM rapamycin and 10 μM chloroquine treated cells can be used as positive control
Downstream tips
Incase of Low CYTO-ID 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 staining, it could be that the cell culture medium was depleted of nutrients and so change media 4-8 hours before the experiment
Anti-LC3 mAb

MBL international corporation

Protocol tips
The LC3-II / LC3-I ratio was 10- to 40-fold lowerr in rapamycin-non treated cells.

Dilute primary at 1:1000
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