Quantitative detection of autophagosomal formation and degradation using flow cytometry. (a) Western blot analysis of wild-type cells, exposed to full medium (FM) or nutrient deprivation (ND) ± bafilomycin A1 (Baf) for 6 h. Cell lysates were analyzed for microtubule-associated protein 1 light chain 3 B (LC3) and β-actin. Quantified bands are expressed relative to FM. (b) Representative image of cells stably expressing mCherry-green fluorescent protein (GFP)-LC3B (tandem-LC3), exposed to FM/ND medium for 6 h ± Baf. (b)i Fluorescent labeling of autophagosomes using tandem-LC3 allows differentiation between autophagosomes, autolysosome formation and degradation. (c-f) Tandem-LC3 cells were exposed to FM/ND ± Baf for 1 to 16 h and fluorescence intensities were analyzed by flow cytometry. Histograms represent distribution of fluorescence intensities of GFP (c) or mCherry (e) after 6 h (upper rows) or 16 h (lower rows) (see Additional file 1 for histograms of FM + Baf). Diagrams show mean fluorescence intensities (relative to fluorescence intensity under FM = 1) of GFP (d) and mCherry (f) after exposure to FM or ND ± Baf for 1 to 16 h.
Hundeshagen et al. BMC Biology 2011 9:38 doi:10.1186/1741-7007-9-38