Autophagy assays

Munafo DB, Colombo MI. A novel assay to study autophagy regulation of autophagosome vacuole size by amino acid deprivation. J Cell Sci 2001 114(Pt 20) 3619-3629. 

AUTONOMOUS CATALYTIC DOMAIN AUTOPHAGY AUTOPHOSPHORYLATION AUTOPROTOLYSIS AUTOPROTOLYSIS CONSTANT Auxiliary enzyme assays,... 

Phospholipidosis (e.g., Nile red, lysotracker dyes, electron microscopy of lysosomal multilamellar bodies), vacuolization, autophagy, lysosomal uptake assays for cell viability (e.g., neutral red)... 

To sum up, an HTS assay including 50 729 compounds has enabled the detection of three autophagy activators that may serve as a starting point for the synthesis of more potent and selective modulators of autophagy. 

Besides multiple staining protocols, a mitochondria potential assay was applied to further assess the toxicity, which was able to rapidly measure the effect of compound on mitochondria potential. After cells were treated with compounds for 60 min, the mitochondria-active dye JC-10 was added for 15-30 min incubation with reagents. In addition, the assay was able to test the effects on inducing autophagy and phospholipidosis after treating the cell with compounds for 24 and 48 h, respectively. The method is compatible for screening of hundreds of compounds within 72 h. 

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