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Superoxide Measurement   

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Abstract

Superoxide dismutase (SOD)-inhibitable reduction of tetrazolium salt, WST-1, is used to measure extracellular superoxide free radical release from various immune cells such as macrophage, neutrophils, microglia (brain macrophage) after stimulation. This protocol has been developed and improved over the years by various researchers in Dr. Hong’s lab, especially Dr. Bin Liu.

Materials and Reagents

  1. Phenol red-free HBSS (Life Technologies, InvitrogenTM, catalog number: 14175-095 )
  2. Superoxide dismutase from bovine erythrocytes (Sigma-Aldrich, catalog number: S7571-30KU )
  3. Phorbol 12-myristate 13-acetate (PMA)
  4. Lipopolysaccharide (LPS)
  5. Fresh WST-1 solution (see Recipes)
  6. SOD stock solution (see Recipes)

Equipment

  1. Microplate spectrophotometer (SpectraMax Plus)
  2. 96-well plates

Procedure

  1. Warm up the media or HBSS to 37 °C.
  2. Turn on the SpectraMax Plus microplate spectrophotometer, set the temperature to 37 °C.
  3. Set up the template for later reading according to your treatment paradigm (this step can also be done later during pretreatment of the cultures with drugs).
  4. Pretreat neuron-glia cultures, macrophages, or microglia grown in 96-well plates with desirable reagents in 100 μl phenol red-free treatment medium or HBSS for 30-60 min.
  5. Add PMA (a positive control), LPS (a common inflammogen), or vehicle in 50 μl of phenol red-free medium/HBSS to the cultures.
  6. Prepare fresh WST-1 solution in the darkroom, protecting the tube from light by covering it with foil.
  7. Add 50 μl of WST-1 (final concentration is 1 mM) in phenol red-free treatment medium/HBSS with or without 600 U ml-1 SOD to the cultures in the dark.
  8. The absorbance at 450 nm is read with a SpectraMax Plus microplate spectrophotometer.
    Note: Read multiple times to observe the dynamic changes of the absorbance. Suggested read time points: 0, 5, 10, 15, 20, 30, 45, 60, 90 min.

Recipes

  1. SOD stock solution
    Dissolve 30 KU of superoxide dismutase from bovine erythrocytes in 1 ml HBSS; store aliquots (100 μl) at -20 °C for later use.
  2. WST-1 solution (fresh, prepare right before use)

References

  1. Gao, H. M., Zhou, H., Zhang, F., Wilson, B. C., Kam, W. and Hong, J. S. (2011). HMGB1 acts on microglia Mac1 to mediate chronic neuroinflammation that drives progressive neurodegeneration. J Neurosci 31(3): 1081-1092.
  2. Zhang, F., Shi, J. S., Zhou, H., Wilson, B., Hong, J. S. and Gao, H. M. (2010). Resveratrol protects dopamine neurons against lipopolysaccharide-induced neurotoxicity through its anti-inflammatory actions. Mol Pharmacol 78(3): 466-477.
Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Gao, H. (2011). Superoxide Measurement. Bio-protocol Bio101: e155. DOI: 10.21769/BioProtoc.155.
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