Autophagy assay cell type - Ramos

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 lysate was centrifuged at 12,000 rpm for 10 min at 4°C. Equivalent amount protein in each sample was separated in SDS-PAGE gel and electro-transferred onto polyvinylidene fluoride membranes. PVDF membranes were incubated with primary antibody and second antibody, respectively. Immobilon™ Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA) was used to visualize the immunoreactive bands, and densitometric values were quantified by ImageJ 1.47v (National Institutes of Health, USA).
Upstream tips
50nM Rapamycin treated cells can be used as positive control.
Protocol tips
Incubate cells for 30 minutes at 37°C in the dark, wash with 1× assay buffer, and resuspend in 500 μL fresh 1× assay buffer. Subject the cells to flow cytometric analysis using the green (FL1) channel.
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