Autophagy assay cell type - HeLa

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 5 matching solutions for this experiment

Atg7 (D12B11) Rabbit mAb

Cell Signaling Technology

Upstream tips
Lyse cells in buffer containing 1.0% (vol/vol) Nonidet P40, 20 mM Tris-HCl, pH 8.0, 10%(vol/vol) glycerol, 150 mM NaCl, 0.2 mM Na3VO4, 1 mM NaF, 0.1 mM sodium pyrophosphate and a protease inhibitor ‘cocktail’
Protocol tips
Dilute primary Ab at 1:100 and incubate at 4 °C for 3 h or overnight.
LC3B (D11) XP® Rabbit mAb

Cell Signaling Technology

Protocol tips
Dilute primary Ab at 1:200 and incubate overnight at 4 °C
Protocol tips
Dilute primary Ab at 1:5000 and incubate overnight
Protocol tips
Dilute primary Ab at 1:1000
Protocol tips
Dilute primary Ab at 1:1000
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