Method to Detect the Cellular Source of Over-Activated NADPH Oxidases Using NAD(P)H Fluorescence Lifetime Imaging

Curr Protoc Cytom. 2017 Apr 3:80:9.52.1-9.52.14. doi: 10.1002/cpcy.20.

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.

Keywords: NADH/NADPH metabolism; NADPH oxidases; fluorescence-lifetime imaging; time-correlated single-photon counting; two-photon microscopy.

MeSH terms

  • Animals
  • Cell Separation
  • Cell Survival
  • Flow Cytometry
  • Fluorescence
  • Humans
  • Imaging, Three-Dimensional / methods*
  • Magnetics
  • Mice
  • NADP / metabolism*
  • NADPH Oxidases / metabolism*
  • Time Factors

Substances

  • NADP
  • NADPH Oxidases