title: Analysis of autophagosome formation using lentiviral biosensors for live fluorescent cellular imaging.
abstract: Autophagy, a highly regulated homeostatic degradative process, allows cells to reallocate nutrients from less important to more essential processes under extreme conditions of starvation. Autophagy also prevents the buildup of damaged proteins and organelles that cause chronic tissue damage and disease. Although a topic of great interest with involvement of multiple signaling pathways, there are limitations in real-time detection of the autophagic process. EMD Millipore has developed technologies where prepackaged, ready-to-use, high-titer lentiviral particles, "lentiviral biosensors," encoding GFP- or RFP-tagged proteins provide a convenient and robust solution for fluorescent imaging of cells undergoing autophagy. Compared to nonviral transfection methods, lentiviral transduction, in many cases, offers higher transfection efficiency and more homogeneous protein expression, particularly for traditionally hard-to-transfect primary cell types. Lentiviral biosensors are ideal for use with fixed and live cell fluorescent microscopy, and are nondisruptive towards cellular function. GFP- or RFP-protein localization matches well with antibody-based immunostaining and demonstrates altered patterns of expression upon treatment with modulators of cell function and phenotype. Lentiviral biosensors provide a broadly effective, convenient method for visualization of cell behavior under a variety of physiological and pathological treatment conditions, in both endpoint and real-time imaging modalities. In this study, we focus on lentiviral biosensors containing GFP-LC3 and RFP-LC3 to study the formation of autophagosomes.
authors.affiliation: EMD Millipore, 28820 Single Oak Drive, Temecula, CA, 92590, USA, Kevin.long@emdmillipore.com.
authors.forename: Kevin
authors.initials: K
authors.lastname: Long
authors.forename: Chandra
authors.initials: C
authors.lastname: Mohan
authors.forename: Janet
authors.initials: J
authors.lastname: Anderl
authors.forename: Karyn
authors.initials: K
authors.lastname: Huryn-Selvar
authors.forename: Haizhen
authors.initials: H
authors.lastname: Liu
authors.forename: Kevin
authors.initials: K
authors.lastname: Su
authors.forename: Mark
authors.initials: M
authors.lastname: Santos
authors.forename: Matthew
authors.initials: M
authors.lastname: Hsu
authors.forename: Lucas
authors.initials: L
authors.lastname: Armstrong
authors.forename: Jun
authors.initials: J
authors.lastname: Ma
mesh_terms: D000818:Animals; D001343:Autophagy; D015374:Biosensing Techniques; D005434:Flow Cytometry / Q000379:methods; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging / Q000295:instrumentation / Q000379:methods*; D014162:Transfection
chemical_list: D049452:Green Fluorescent Proteins