Autophagy assay cell type - REH

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 4 matching solutions for this experiment

MAP LC3β Antibody

Santa Cruz Biotechnology

Upstream tips
MAP LC3β Antibody (G-9) is a mouse monoclonal IgG2b (kappa light chain)
Protocol tips
1:300 dilution of primary antibody and incubation for 1 hour at RT
Protocol tips
Time Kinetics experiments are good to observe the optimal Cyto-ID staining (different time points between 6 to 24 hrs).
Protocol tips
rest cells for 2 h and then treat with absence or presence of the autophagy inhibitor (5 mM, 3-MA)
Protocol tips
10nM of Bafilomycin to treat the cells.
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