Skip to main content
NCBI home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1992 Aug;30(8):2071–2076. doi: 10.1128/jcm.30.8.2071-2076.1992

Rapid whole-blood microassay using flow cytometry for measuring neutrophil phagocytosis.

C White-Owen 1, J W Alexander 1, R M Sramkoski 1, G F Babcock 1
PMCID: PMC265444  PMID: 1500514

Abstract

A simple flow cytometric method (FCM) for measuring phagocytosis of Staphylococcus aureus by human neutrophils (polymorphonuclear leukocytes [PMNs]) is described. This assay utilizes 100 microliters of EDTA-anticoagulated whole blood and a simplified method of fluorescently labeling bacteria. A commercially available whole-blood lysing reagent allows for the removal of erythrocytes and the exclusion of external free or adherent bacteria. Phagocytized bacteria are unaffected by this reagent, so PMNs containing internalized bacteria can be easily identified by FCM. Advantages of this method include the following: (i) small sample size, (ii) no requirement for PMN separation, (iii) rapid reliable method of labeling the bacteria, (iv) ability to distinguish between adherent bacteria and those which are actually internalized, (v) avoidance of vital dyes as quenching agents, and (vi) ability to fix cells and store for future FCM analysis.

Full text

PDF
2071

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bassøe C. F., Bjerknes R. Phagocytosis by human leukocytes, phagosomal pH and degradation of seven species of bacteria measured by flow cytometry. J Med Microbiol. 1985 Feb;19(1):115–125. doi: 10.1099/00222615-19-1-115. [DOI] [PubMed] [Google Scholar]
  2. Bassøe C. F., Laerum O. D., Glette J., Hopen G., Haneberg B., Solberg C. O. Simultaneous measurement of phagocytosis and phagosomal pH by flow cytometry: role of polymorphonuclear neutrophilic leukocyte granules in phagosome acidification. Cytometry. 1983 Nov;4(3):254–262. doi: 10.1002/cyto.990040311. [DOI] [PubMed] [Google Scholar]
  3. Bjerknes R., Bassøe C. F. Human leukocyte phagocytosis of zymosan particles measured by flow cytometry. Acta Pathol Microbiol Immunol Scand C. 1983 Oct;91(5):341–348. [PubMed] [Google Scholar]
  4. Bjerknes R., Bassøe C. F., Sjursen H., Laerum O. D., Solberg C. O. Flow cytometry for the study of phagocyte functions. Rev Infect Dis. 1989 Jan-Feb;11(1):16–33. doi: 10.1093/clinids/11.1.16. [DOI] [PubMed] [Google Scholar]
  5. Bjerknes R. Flow cytometric assay for combined measurement of phagocytosis and intracellular killing of Candida albicans. J Immunol Methods. 1984 Aug 3;72(1):229–241. doi: 10.1016/0022-1759(84)90451-4. [DOI] [PubMed] [Google Scholar]
  6. Cantinieaux B., Hariga C., Courtoy P., Hupin J., Fondu P. Staphylococcus aureus phagocytosis. A new cytofluorometric method using FITC and paraformaldehyde. J Immunol Methods. 1989 Jul 26;121(2):203–208. doi: 10.1016/0022-1759(89)90161-0. [DOI] [PubMed] [Google Scholar]
  7. Fattorossi A., Nisini R., Pizzolo J. G., D'Amelio R. New, simple flow cytometry technique to discriminate between internalized and membrane-bound particles in phagocytosis. Cytometry. 1989 May;10(3):320–325. doi: 10.1002/cyto.990100311. [DOI] [PubMed] [Google Scholar]
  8. Gelfand J. A., Fauci A. S., Green I., Frank M. M. A simple method for the determination of complement receptor-bearing mononuclear cells. J Immunol. 1976 Mar;116(3):595–599. [PubMed] [Google Scholar]
  9. Hasui M., Hirabayashi Y., Kobayashi Y. Simultaneous measurement by flow cytometry of phagocytosis and hydrogen peroxide production of neutrophils in whole blood. J Immunol Methods. 1989 Feb 8;117(1):53–58. doi: 10.1016/0022-1759(89)90118-x. [DOI] [PubMed] [Google Scholar]
  10. Hed J., Hallden G., Johansson S. G., Larsson P. The use of fluorescence quenching in flow cytofluorometry to measure the attachment and ingestion phases in phagocytosis in peripheral blood without prior cell separation. J Immunol Methods. 1987 Jul 16;101(1):119–125. doi: 10.1016/0022-1759(87)90224-9. [DOI] [PubMed] [Google Scholar]
  11. Ogle J. D., Ogle C. K., Noel J. G., Hurtubise P., Alexander J. W. Studies on the binding of C3b-coated microspheres to human neutrophils. J Immunol Methods. 1985 Jan 21;76(1):47–62. doi: 10.1016/0022-1759(85)90480-6. [DOI] [PubMed] [Google Scholar]
  12. Seeds M. C., Parce J. W., Szejda P., Bass D. A. Independent stimulation of membrane potential changes and the oxidative metabolic burst in polymorphonuclear leukocytes. Blood. 1985 Jan;65(1):233–240. [PubMed] [Google Scholar]
  13. Verhoef J., Waldvogel F. A. Testing phagocytic cell function. Eur J Clin Microbiol. 1985 Aug;4(4):379–381. doi: 10.1007/BF02148686. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES