Autophagy assay cell type - AML12

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

CYTO-ID® Autophagy detection kit

Enzo Life Sciences

Upstream tips	Protocol tips	Downstream tips
	- Add reagent and incubated cells for 30 minutes at 37°C in the dark	- 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
- Add reagent and incubated cells for 30 minutes at 37°C in the dark
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.

Manufacturer protocol Publication protocol 1 Relevant paper

SQSTM1 monoclonal antibody (M01), clone 2C11

Abnova

Upstream tips	Protocol tips	Downstream tips
	- primary antibody dilution immunostaining (1:100 dilution) and immunoblotting (1:2000 dilution). - incubate with the primary antibody at 4 °C overnight

Protocol tips
- primary antibody dilution immunostaining (1:100 dilution) and immunoblotting (1:2000 dilution). - incubate with the primary antibody at 4 °C overnight

Manufacturer protocol Publication protocol 1 Relevant paper

Anti-LC3B antibody produced in rabbit

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
	- Use a working concentration of 1-2 mg/mL. - Incubate primary antibody overnight at 4C.

Protocol tips
- Use a working concentration of 1-2 mg/mL. - Incubate primary antibody overnight at 4C.

Manufacturer protocol Publication protocol 1 Relevant paper

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