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

Daniel Bremer1, Ruth Leben2, Ronja Mothes1, Helena Radbruch1, Raluca Niesner2

1 Neuropathology Department, Charité – Universitätsmedizin, Berlin, 2 Biophysical Analytics, German Rheumatism Research Center, Leibniz Institute, Berlin
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.52
DOI:  10.1002/cpcy.20
Online Posting Date:  April, 2017
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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: fluorescence‐lifetime imaging; NADPH oxidases; NADH/NADPH metabolism; two‐photon microscopy; time‐correlated single‐photon counting

     
 
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Table of Contents

  • Introduction
  • Strategic Statement
  • Basic Protocol 1: Generate Time‐domain NAD(P)H FLIM Data of Living Cells
  • Basic Protocol 2: Exponential Analysis of the NAD(P)H‐FLIM Data
  • Support Protocol 1: Cell Isolation by MACS Miltenyi Biotec
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Generate Time‐domain NAD(P)H FLIM Data of Living Cells

  Materials
  • Ιsolated cells (see Support Protocol) in medium
  • Heated liquid mixture of bee wax and petrolatum (Vaseline)
  • Glass slides
  • Rectangular coverslips
  • 37°C, 5% CO 2 incubator
  • Multi‐photon microscope equipped with a high resolution objective lens (NA ≈ 1), in our case, TriMScope II, (LaVision Biotec)
  • Heating plate for the cells (heating foil)
  • Time‐correlated single‐photon counting detector (TCSPC), in our case 16 × ‐FLIM detector (LaVision Biotec)
  • Heating foil for the objective lens

Basic Protocol 2: Exponential Analysis of the NAD(P)H‐FLIM Data

  Materials
  • Raw‐data decay image stacks
  • ImageJ/Fiji
  • Python (with Python Imaging Library: PIL and numPy/sciPy packages)

Support Protocol 1: Cell Isolation by MACS Miltenyi Biotec

  Materials
  • Μurine spleen or blood
  • MACs buffer (see recipe)
  • B Cell Isolation kit (Miltenyi Biotec, cat. no. 130-090-862) containing:
    • Biotin‐Antibody cocktail
  • CD11b Microbeads, human and mouse (Miltenyi Biotec, cat. no. 130‐049‐601)
  • Phenol‐free RPMI medium
  • 30‐μm nylon mesh
  • Centrifuge
  • MACS columns and MACS Separators: Choose the appropriate MACS Separator and MACS Columns according to the number of labeled cells and to the number of total cells
  • Pre‐separation filters (20 µm)
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Figures

Videos

Literature Cited

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  Mossakowski, A. A., Pohlan, J., Bremer, D., Lindquist, R., Millward, J. M., Bock, M., … Radbruch, H. (2015). Tracking CNS and systemic sources of oxidative stress during the course of chronic neuroinflammation. Acta Nuropathologica, 130, 799–814. doi: 10.1007/s00401‐015‐1497‐x
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  Niesner, R., Peker, B., Schlusche, P., & Gericke, K. H. (2004). Noniterative biexponential fluorescence lifetime imaging in the investigation of cellular metabolism by means of NAD(P)H autofluorescence. Chemphyschem: A European Journal of Chemical Physics and Physical Chemistry, 5, 1141–1149. doi: 10.1002/cphc.200400066
  Niesner, R., Narang, P., Spiecker, H., Andresen, V., Gericke, K. H., & Gunzer, M. (2008). Selective Detection of NADPH Oxidase in Polymorphonuclear Cells by Means of NAD(P)H‐Based Fluorescence Lifetime Imaging. Journal of Biophysics, 2008. doi: 10.1155/2008/602639
  Radbruch, H., Bremer, D., Guenther, R., Cseresnyes, Z., Lindquist, R., Hauser, A. E., & Niesner, R. (2016). Ongoing Oxidative Stress Causes Subclinical Neuronal Dysfunction in the Recovery Phase of EAE. Frontiers in Immunology, 7, 92. doi: 10.3389/fimmu.2016.00092
  Stringari, C., Nourse, J. L., Flanagan, L. A., & Gratton, E. (2012). Phasor fluorescence lifetime microscopy of free and protein‐bound NADH reveals neural stem cell differentiation potential. PloS One, 7, e48014. doi: 10.1371/journal.pone.0048014
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  Wahl, M., Rahn, H. J., Rohlicke, T., Kell, G., Nettels, D., Hillger, F., … Erdmann, R. (2008). Scalable time‐correlated photon counting system with multiple independent input channels. The Review of Scientific Instruments, 79, 123113. doi: 10.1063/1.3055912
Key References
  Niesner, R., Narang, P., Spiecker, H., Andresen, V., Gericke, K. H., & Gunzer, M. (2008). Selective Detection of NADPH Oxidase in Polymorphonuclear Cells by Means of NAD(P)H‐Based Fluorescence Lifetime Imaging. Journal of Biophysics, 2008, 602639. doi: 10.1155/2008/602639.
  Mossakowski, A. A., Pohlan, J., Bremer, D., Lindquist, R., Millward, J. M., Bock, M., … Radbruch, H. (2015). Tracking CNS and systemic sources of oxidative stress during the course of chronic neuroinflammation. Acta Neuropathologica, 130, 799–814. doi: 10.1007/s00401‐015‐1497‐x
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