Oxidative Metabolism

J. Paul Robinson1

1 Purdue University Cytometry Laboratories, West Lafayette, Indiana
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.7
DOI:  10.1002/0471142956.cy0907s02
Online Posting Date:  May, 2001
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Abstract

Peroxide production is detected using DCFH‐DA, superoxide anion by hydroethidine; the methods can be combined for simultaneous detection. A support protocol describes calibration of the flow cytometer to permit calculation of the actual H2O2 production per cell. These protocols are applicable to many different cell types, but provide detailed information on using neutrophils, highly reactive cells that produce vast quantities of ROS.

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of H2O2 Production in the Oxidative Burst of Human Neutrophils Using DCFH‐DA
  • Alternate Protocol 1: Measurement of Superoxide (O2−) Production in Neutrophils Using Hydroethidine
  • Alternate Protocol 2: Simultaneous Measurement of H2O2 AND O2−In Neutrophils
  • Alternate Protocol 3: Reactive Oxygen Metabolism Using Dihydrorhodamine 123
  • Support Protocol 1: Calibration of the Flow Cytometer for H2O2Measurements
  • Support Protocol 2: Preparation of Vacutainer Tubes with Preservative‐Free Heparin
  • Support Protocol 3: Preparation of PBS‐Gel
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measurement of H2O2 Production in the Oxidative Burst of Human Neutrophils Using DCFH‐DA

  Materials
  • Vacutainer tubes treated with preservative‐free heparin (see protocol 6)
  • Ammonium chloride lysing solution (working solution; appendix 2A)
  • Hanks balanced salt solution (HBSS; appendix 2A)
  • 20 mM 2′,7′‐dichlorofluorescin diacetate (DCFH‐DA, from Molecular Probes; prepare fresh in 100% ethanol and keep on ice, protected from light)
  • 10 µg/ml phorbol myristate acetate (PMA) working solution I (see recipe)
  • 50‐ml centrifuge tubes, sterile
  • 12 × 75–mm polyethylene tube and caps

Alternate Protocol 1: Measurement of Superoxide (O2−) Production in Neutrophils Using Hydroethidine

  • Cells in culture (optional)
  • 10 mM hydroethidine (HE, from Polysciences; mol. wt. 315; prepare fresh in DMSO and keep on ice, protected from light)

Alternate Protocol 2: Simultaneous Measurement of H2O2 AND O2−In Neutrophils

  • 10 mM hydroethidine (HE, Polysciences; MW 315) in DMSO (prepare fresh and keep on ice, protected from light)

Alternate Protocol 3: Reactive Oxygen Metabolism Using Dihydrorhodamine 123

  • 50 mM dihydrorhodamine 123 (DHR; see recipe)
  • 3 mM propidium iodide (PI) solution (see recipe)
  • 100 µM formyl‐methionyl‐leucyl‐phenylalanine (fMLP) working solution (see recipe)

Support Protocol 1: Calibration of the Flow Cytometer for H2O2Measurements

  • Absolute ethanol
  • 200 ng/ml phorbol myristate acetate (PMA) working solution II (see recipe)
  • PBS‐gel (see protocol 7)
  • Reagent‐grade dichlorofluorescein (DCF; Sigma)
  • Sonicator
  • 10‐ to 20‐ml polycarbonate centrifuge tubes
  • Statistical software for correlation

Support Protocol 2: Preparation of Vacutainer Tubes with Preservative‐Free Heparin

  Materials
  • 16 × 100–mm red‐top Vacutainer tubes
  • 2‐ml bottle of 1000 U/ml preservative‐free heparin (LyphoMed)
  • Alcohol wipes
  • 1.0‐ml allergist syringes

Support Protocol 3: Preparation of PBS‐Gel

  Materials
  • Disodium EDTA
  • Glucose
  • Gelatin (Difco)
  • PBS ( appendix 2A)
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Figures

Videos

Literature Cited

Literature Cited
   Bass, D.A., Parce, J.W., De Chatelet, L.R., Szejda, P., Seeds, M.C., and Thomas, M. 1983. Flow cytometric studies of oxidative product formation by neutrophils: A graded response to membrane stimulation. J. Immunol. 130:1910‐1917.
   Duque, R.E., Robinson, J.P., Hudson, J.L., Till, G.O., and Ward, P.A. 1985. Fed. Proc. (abstr.).
   Loesche, W.J., Robinson, J.P., Flynn, M., Hudson, J.L., and Duque, R.E. 1988. Infect. Immun. 56:156‐160.
   Robinson, J.P., Bruner, L.H., Bassøe, C.F., Hudson, J.L., Ward, P.A., and Pham, S.H. 1983. Measurement of intracellular fluorescence on human monocytes relative to oxidative metabolism. J. Leukocyte Biol. 43:304‐310.
   Rothe, G. and Valet, G. 1990. Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2′,7′‐dichlorofluorescin. J. Leukocyte Biol. 47:440‐448.
   Rothe, G., Oser, A., and Valet, G. 1988. Dihydrorhodamine 123: A new flow cytometric indicator for respiratory burst activity in neutrophil granulocytes. Naturwissenschaften 75:354‐355.
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