Autophagy assay cell type - SP53

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
This dye specifically accumulates in the autophagosomes allowing the evaluation of the extent of autophagy as number of green fluorescent spots into each single cell.
Protocol tips
Use Cyto-ID green dye (1 μl/4 ml assay buffer) for 30 minutes in dark at RT
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.
Atg5 Antibody

Cell Signaling Technology

Protocol tips
Incubate the membrane in primary antibody solution for 1 hour at room temperature or overnight at 4˚C with gentle rocking. This time may require optimization. In most cases, overnight incubation at 4˚C increases signal strength and reduces background signal relative to 1 hour incubation at room temperature.
Protocol tips
Dilute primary antibody in 1% normal serum or BSA (in 1X PBS). Incubate coverslips with ~5 µ g/mL rabbit anti-LC3 primary antibody for 1 hour at room temperature (37°C is optional), or for 16 hours at 4°C.
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