Autophagy assay cell type - GL261

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

LC3B (D11) XP® Rabbit mAb (Biotinylated)

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
Lyse cells in lysis buffer (1% triton-X, 0.1% SDS, 50 mM HEPES pH 7.4, 150 mM NaCl, 100 mM NaF, 10 mM EDTA, 10 mM Na4P2O7 containing protease inhibitor cocktail )	Dilute primary Ab at 1:1000

Upstream tips
Lyse cells in lysis buffer (1% triton-X, 0.1% SDS, 50 mM HEPES pH 7.4, 150 mM NaCl, 100 mM NaF, 10 mM EDTA, 10 mM Na4P2O7 containing protease inhibitor cocktail )
Protocol tips
Dilute primary Ab at 1:1000

Manufacturer protocol Publication protocol 1 Relevant paper

mTOR Inhibitor XI, Torin1 - Calbiochem

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
Highly potent inhibitor for mTOR and DNA-PK while inhibits PI3-K only at much higher concentration	250nM of Torin1 to treat the cells

Upstream tips
Highly potent inhibitor for mTOR and DNA-PK while inhibits PI3-K only at much higher concentration
Protocol tips
250nM of Torin1 to treat the cells

Manufacturer protocol Publication protocol 1 Relevant paper

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