Autophagy assay cell type - L929

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 3 matching solutions for this experiment

LC3B Antibody Kit for Autophagy

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
Use the cells pretreated with 30 µM chloroquine disphosphate for 16 h as a positive control.	Incubate the fixed and permeabilized cells with primary antibody for 1hr followed by 1hr incubation with secondary antibody.

Upstream tips
Use the cells pretreated with 30 µM chloroquine disphosphate for 16 h as a positive control.
Protocol tips
Incubate the fixed and permeabilized cells with primary antibody for 1hr followed by 1hr incubation with secondary antibody.

Manufacturer protocol Publication protocol 1 Relevant paper

Dansylcadaverine

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
MDC is an autofluorescent agent used as selective marker for autophagic vacuoles and especially for autolysosomes.	Stain the cells with 0.05 mmol/L MDC at 37oC for 1 h.

Upstream tips
MDC is an autofluorescent agent used as selective marker for autophagic vacuoles and especially for autolysosomes.
Protocol tips
Stain the cells with 0.05 mmol/L MDC at 37oC for 1 h.

Manufacturer protocol Publication protocol 1 Relevant paper

Anti-LC3 antibody produced in rabbit

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
Lyse cells in Laemmli SDS buffer	Incubate primary Ab with agitation for 1 hour. The solution should move freely across the surface of the membrane.

Upstream tips
Lyse cells in Laemmli SDS buffer
Protocol tips
Incubate primary Ab with agitation for 1 hour. The solution should move freely across the surface of the membrane.

Manufacturer protocol Publication protocol 1 Relevant paper

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