Autophagy assay cell type - Mel cells

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
Kit contains specific dye that selectively stains autophagic compartments. This kit uses a green fluorescent probe that reacts with autophagic vacuoles and is detectable by flow cytometry		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 experimen

Upstream tips
Kit contains specific dye that selectively stains autophagic compartments. This kit uses a green fluorescent probe that reacts with autophagic vacuoles and is detectable by flow cytometry
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 experimen

Manufacturer protocol Publication protocol 1 Relevant paper

Torin 1

Tocris

Upstream tips	Protocol tips	Downstream tips
Potent and selective mTOR inhibitor that Displays 200-fold selectivity for mTOR over DNA-PK, ATM and hVps34	2nM and 10nM concentration of Torin1 to be used to induce autophagy.

Upstream tips
Potent and selective mTOR inhibitor that Displays 200-fold selectivity for mTOR over DNA-PK, ATM and hVps34
Protocol tips
2nM and 10nM concentration of Torin1 to be used to induce autophagy.

Manufacturer protocol Publication protocol 1 Relevant paper

LC3A/B (D3U4C) XP® Rabbit mAb #12741

Cell Signaling Technology

Upstream tips	Protocol tips	Downstream tips
	1:1000 dilution of Primary antibody and incubate 1 h overnight

Protocol tips
1:1000 dilution of Primary antibody and incubate 1 h overnight

Manufacturer protocol Publication protocol 1 Relevant paper

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