title: Method to Detect the Cellular Source of Over-Activated NADPH Oxidases Using NAD(P)H Fluorescence Lifetime Imaging.
abstract: Fluorescence-lifetime imaging microscopy (FLIM) is a technique to generate images, in which the contrast is obtained by the excited-state lifetime of fluorescent molecules instead of their intensity and emission spectrum. The ubiquitous coenzymes NADH and NADPH, hereafter NAD(P)H, in cells show a short fluorescence lifetime ≈400 psec in the free-state and a longer fluorescence lifetime when bound to enzymes. The fluorescence lifetime of NAD(P)H in this state depends on the binding-site on the specific enzyme. In the case of NADPH bound to members of the NADPH oxidases family we measured a fluorescence lifetime of 3650 psec as compared to enzymes typically active in cells, in which case fluorescence lifetimes of ∼2000 psec are measured. Here we present a robust protocol based on NAD(P)H fluorescence lifetime imaging in isolated cells to distinguish between normally active enzymes and NADPH oxidases, mainly responsible for oxidative stress. © 2017 by John Wiley & Sons, Inc.
authors.affiliation: Biophysical Analytics, German Rheumatism Research Center, Leibniz Institute, Berlin, Germany.|Neuropathology Department, Charité - Universitätsmedizin, Berlin, Berlin, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Bremer
authors.affiliation: Biophysical Analytics, German Rheumatism Research Center, Leibniz Institute, Berlin, Germany.
authors.forename: Ruth
authors.initials: R
authors.lastname: Leben
authors.affiliation: Biophysical Analytics, German Rheumatism Research Center, Leibniz Institute, Berlin, Germany.|Neuropathology Department, Charité - Universitätsmedizin, Berlin, Berlin, Germany.
authors.forename: Ronja
authors.initials: R
authors.lastname: Mothes
authors.affiliation: Neuropathology Department, Charité - Universitätsmedizin, Berlin, Berlin, Germany.
authors.forename: Helena
authors.initials: H
authors.lastname: Radbruch
authors.affiliation: Biophysical Analytics, German Rheumatism Research Center, Leibniz Institute, Berlin, Germany.
authors.forename: Raluca
authors.initials: R
authors.lastname: Niesner
mesh_terms: D000818:Animals; D002469:Cell Separation; D002470:Cell Survival; D005434:Flow Cytometry; D005453:Fluorescence; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008280:Magnetics; D051379:Mice; D009249:NADP / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism*; D013997:Time Factors
chemical_list: D009249:NADP; D019255:NADPH Oxidases
keywords: NADH/NADPH metabolism; NADPH oxidases; fluorescence-lifetime imaging; time-correlated single-photon counting; two-photon microscopy