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Inflammation

, Volume 8, Issue 3, pp 287–300 | Cite as

Activated rat neutrophils

A sequential quantitative assay for aggregation and NBT reduction
  • Kenneth O. Fennell
  • Howard R. Creamer
  • Walter L. Gabler
  • Arthur C. Brown
  • Wesley W. Bullock
Original Articles

Abstract

We present here a rapid, sensitive, and convenient approach for the analysis of activated Lewis rat PMNs based on detecting separately, or in tandem, PMN aggregation and PMN reduction of nitroblue tetrazolium (NBT). These responses are quantitated using an ELISA scanner which can rapidly measure optical densities of cell cultures in microtiter plates. Aggregation induced by as little as 0.005 (Μg/ml of phorbol myristate acetate (PMA), 0.01 Μg/ml lipopolysaccharide (LPS), or a 1∶160 dilution of lymphokine-containing rat serum can be detected employing this approach. NBT reduction was induced by as little as 0.01 Μg/ml PMA. Blocking studies employing 2-deoxyglucose, iodoacetamide, and polymyxin B gave the expected results and confirmed that these assays detect cellular responses to soluble stimuli. Using this technology the effects of PMA and LPS on rat peritoneal exudate PMNs were evaluated. Rat PMNs appeared less sensitive to LPS than human PMNs and also reduced NBT more slowly following stimulation with PMA. Because of the slowness in NBT reduction following stimulation, NBT reduction can be evaluated, in tandem, after measuring aggregation. The simplicity of this system, coupled with the speed with which large numbers of mirocultures can be read and the low number of cells required, make this approach for studying responses especially attractive.

Keywords

Tetrazolium Phorbol Myristate Acetate Nitroblue Tetrazolium Myristate Polymyxin 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Plenum Publishing Corporation 1984

Authors and Affiliations

  • Kenneth O. Fennell
    • 1
  • Howard R. Creamer
    • 1
  • Walter L. Gabler
    • 1
  • Arthur C. Brown
    • 1
  • Wesley W. Bullock
    • 1
  1. 1.Schools of Dentistry and Medicine Department of Microbiology, Biochemistry, PhysiologyOregon Health Sciences UniversityPortland

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