Autophagy assay cell type - Saos

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

Upstream tips
Use 2 μM ATRA treated Saos cells as positive control. ATRA is known to modulate autophagy in this cell line.
Downstream tips
Incase of Low CYTO-ID Green dye staining in all treatments, increase the reagent concentration (500X dilution of the dye is recommended) and the incubation time.

Incase of High CYTO-ID Green staining, it could be that the cell culture medium was depleted of nutrients and so change media 4-8 hours before the experiment.
Protocol tips
Incubate the membrane with appropriate dilutions of primary antibody in blocking buffer and incubate overnight at 4°C;
LC3B Antibody #2775

Cell Signaling Technology

Protocol tips
1:2000 dilution of Primary antibody
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