Autophagy assay cell type - 143B

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

Protocol tips
- Incubate primary antibody overnight at 4 °C
Downstream tips
- If no signal or weak signal or non-specific bands, make sure Enzyme inhibitor (Sodium azide) is not present as it inhibits
Protocol tips
- Stain on ice for 30 minutes in the dark after adding Anti-LC3.
Downstream tips
- The kit is designed to measure endogenous levels of LC3.
Protocol tips
- Stain the cells for 1 hour at 37 C in dark.
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