, 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


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.


Tetrazolium Phorbol Myristate Acetate Nitroblue Tetrazolium Myristate Polymyxin 
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  1. 1.
    Gudewicz, P. W., D. H. Beezhold, V. A. Person, andJ. Molnar. 1982. Lack of Stimulation of post-phagocytic metabolic activities of polymorphonuclear leukocytes by fibronectin opsonized particles.J. Reticuloendothel. Soc. 32:143–154.Google Scholar
  2. 2.
    Wilson, M. E., D. P. Jones, P. Munkenbeck, andD. C. Morrison. 1982. Serum-dependent and independent effects of bacterial lipolysaccharides on human neutrophil oxidative capacity in vitro.J. Reticuloendothel. Soc. 31:43–57.Google Scholar
  3. 3.
    Pick, E., J. Charon, andD. Mizel. 1981. A rapid densitometric microassay for nitroblue tetrazolium reduction and application of the microassay to macraphages.J. Reticuloendothel Soc. 30:581–593.Google Scholar
  4. 4.
    Bullock, W. W., H. R. Creamer, andW. L. Gabler. 1982. A rapid quantitative assay for activated neutrophils.Inflammation 6:305–318.Google Scholar
  5. 5.
    Lowry, O. H., N. J. Rosebrough, A. L. Farr, andR. J. Randall. 1951. Protein measurements with the Folin-phenol reagent.J. Biol. Chem. 193:265–275.Google Scholar
  6. 6.
    Tabachnick, M., andH. Sobotka. 1959. Azoproteins. I. Spectrophotometric studies of amino acid azo derivatives.J. Biol. Chem. 234:1726–1730.Google Scholar
  7. 7.
    Tabachnick, M., andH. Sobotka. 1959. Azoproteins. IL A Spectrophotometric study of the coupling of diazotized arsanilic acid with proteins.J. Biol. Chem. 235:1051–1054.Google Scholar
  8. 8.
    Neta, R., andS. B. Salvin. 1981.In Lymphokines: A Forum for Immunoregulatory Cell Products, Vol. 2. Edgar Pick, editor. Academic Press, New York. 295–308.Google Scholar
  9. 9.
    Boyum, A. 1968. Isolation of mononuclear cells and granulocytes from human blood.Scand. J. Lab. Invest. 21(Suppl. 97):77–89.Google Scholar
  10. 10.
    Tsan, M. F., andR. C. Denison. 1980. Phorbol myristate acetate-induced neutrophil autotoxicity. A comparison with H2O2 toxicity.Inflammation 4:371–380.Google Scholar
  11. 11.
    MaCgregor, R., P. Spagnuolo, andA. Lentner. 1974. Inhibition of granulocyte adherence by ethanol, prednisone, and aspirin measured with an assay system.N. Engl. J. Med. 291:642–645.Google Scholar
  12. 12.
    O'flaherty, J. T.. 1980. Involvement of bivalent cations and arachidonic acid in neutrophil aggregation.Inflammation 4:181–194.Google Scholar
  13. 13.
    Craddock, P. R., D. E. Hammerschmidt, J. G. White, A. P. Dalmasso, andH. S. Jacob. 1977. Complement (C5a)-induced granulocyte aggregation in vitro: A possible mechanism of complement-mediated leukopenia.J. Clin. Invest. 60:260–264.Google Scholar
  14. 14.
    Craddock, P. R., J. G. White, D. J. Weisdorf, andD. E. Hammerschmidt. 1980. Digital integration responses. A simple and reproducible method for the quantitation of granulocyte adhesiveness.Inflammation 4:381–394.Google Scholar
  15. 15.
    Born, G. V. R., andM. Hume. 1967. Effects of the numbers and sizes of platelet aggregates on the optical density of plasma.Nature 215:1027–1029.Google Scholar

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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