Autophagy assay cell type - H4 Human neuroblastoma cells

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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Protocol tips
The silenced cells were treated with or without the autophagy inhibitors BafA1 (Millipore, 196000) or CQ (Sigma-Aldrich, C6628) and then lysed in RIPA buffer (1% NP40, 50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.25% sodium deoxycholate, 1% sodium dodecyl sulfate (SDS), protease inhibitor cocktail and phosphatase inhibitor). The proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes for immunoblotting. The membrane were rinsed with Odyssey Blocking Buffer (Li-COR, 927-50000) and then incubated overnight at 4°C with primary antibodies against ATG4B (A2981), MAP1LC3 (L7543) and ACTB (A5441) (purchased from Sigma-Aldrich); p27kip1 (purchased from Santa Cruz Biotechnology); and CCND1 (cyclin D1, 2926), pThr172-AMPK (5536), AMPK (2532), pSer792-raptor (2083) and raptor (2280) (purchased from Cell Signaling Technology). The proteins were probed with an HRP-labeled secondary antibody (Santa Cruz Biotechnology, sc-2004 or sc-2005) and detected with an ECL reagent. The membrane was scanned and analyzed for protein expression levels using the ChemiDoc XRS Imaging System (Bio-Rad). The protein levels were measured using ImageJ software.
Protocol tips
The silenced cells were treated with or without the autophagy inhibitors BafA1 (Millipore, 196000) or CQ (Sigma-Aldrich, C6628) and then lysed in RIPA buffer (1% NP40, 50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.25% sodium deoxycholate, 1% sodium dodecyl sulfate (SDS), protease inhibitor cocktail and phosphatase inhibitor). The proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes for immunoblotting. The membrane were rinsed with Odyssey Blocking Buffer (Li-COR, 927-50000) and then incubated overnight at 4°C with primary antibodies against ATG4B (A2981), MAP1LC3 (L7543) and ACTB (A5441) (purchased from Sigma-Aldrich); p27kip1 (purchased from Santa Cruz Biotechnology); and CCND1 (cyclin D1, 2926), pThr172-AMPK (5536), AMPK (2532), pSer792-raptor (2083) and raptor (2280) (purchased from Cell Signaling Technology). The proteins were probed with an HRP-labeled secondary antibody (Santa Cruz Biotechnology, sc-2004 or sc-2005) and detected with an ECL reagent. The membrane was scanned and analyzed for protein expression levels using the ChemiDoc XRS Imaging System (Bio-Rad). The protein levels were measured using ImageJ software.
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