What is Autophagy?
Autophagy is the lysosomal degradation route for soluble components of the cytosol and organelles. It helps to remove damaged organelles and misfolded or aggregated proteins. Cytoplasmic components become enclosed in a double membrane to form a compartment known as the autophagosome and are delivered to the lysosome for degradation. Autophagy failure is implicated in development neurodegenerative disease and cancer. Investigation of autophagy process may have applications in neurodegenerative disease and cancer treatment.
How to monitor or visualize autophagy flux?
Most assays for to quantify autophagosome formation use the autophagy marker protein LC3 (microtubule-associated protein 1 light chain 3). In the autophagy flux, LC3 is cleaved (became LC3-II) then tightly bound to the autophagosomal membranes and serves as an autophagic marker protein. Fluorescent labeled LC3B for autophagy is widely used tool to visualize autophagosome formation. By creating fluorescent-LC3 fusion constructs (GFP-LC3 or RFP-LC3), you can visualize autophagy in real time in live cells under fluorescent-microscope. Also, the turnover of Fluorescent-LC3 can be quantify using flow cytometry to measure autophagic activity in living mammalian cells.
Gentarget’s Products for autophagy research
Gentarget developed lentivirus products that express Fluorescent-LC3 fusion constructs, GFP-LC3 (CAT#: LVP399-G), RFP-LC3 (CAT#: LVP399-R) or CFP-LC3 (CAT#: LVP399-C), containing the Puromycin antibiotic selection for stable cell line generation. Those products are used to monitor induction of autophagy after amino acid starvation or rapamycin treatment as well as autophagosome formation
Expression of the ‘Fluorescent-LC3’ fusion allows to visualize autophagosome formation in real time in live cells. During autophagosome formation, the fusion protein is processed and recruited to the autophagosome membrane, where it can be imaged as cytoplasmic puncta by high resolution fluorescence microscopy. The changes in percentage of Fluorescent positive cells can be determined and serves as the indicative of autophagy processing stages.