Autophagy assay cell type - Beas-2B

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

Upstream tips
- Lyse cells in RIPA buffer (2 mM pH 8.0 ETDA, 50 mMTris pH 7.4, 150 mMNaCl, 1% NP-40, 1 mM PMSF, 1% sodium deoxycholate) containing protease inhibitor cocktail
Protocol tips
- Dilute Ab at 1:1000 and incubate overnight at 4°C with primary antibody
Autophagy Assay Kit

Sigma-Aldrich

Protocol tips
- Add autophagosome reagent incubate for 1h at 37 °C, 5% CO2
Protocol tips
- 200 µM concentration of 3-MA can be used to treat the cells
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