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A rapid density gradient technique for separating polymorphonuclear granulocytes

A Cabanis1, B Gressier, S Lebegue

  • 1Faculté des Sciences Pharmaceutiques et Biologiques, Laboratoire de Pharmacologie, Pharmacocinétique et Pharmacie Clinique, Lille, France.

APMIS : Acta Pathologica, Microbiologica, Et Immunologica Scandinavica
|February 1, 1994
PubMed
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A novel gradient separation method effectively isolates pure polymorphonuclear neutrophils (PMNs) from blood. These isolated PMNs demonstrate high reactive oxygen species generation, proving useful for in vitro research.

Area of Science:

  • Hematology
  • Immunology
  • Cell Biology

Background:

  • Accurate isolation of polymorphonuclear neutrophils (PMNs) is crucial for various in vitro immunological and cellular studies.
  • Existing methods for PMN isolation may suffer from low yield, purity issues, or cell activation.
  • Developing an efficient and reliable method for obtaining pure, functional PMNs is essential.

Purpose of the Study:

  • To compare a gradient separation technique with isotonic ammonium chloride haemolysis against two other methods for isolating PMNs from human blood.
  • To evaluate the yield, purity, and functional capacity (reactive oxygen species generation) of PMNs isolated by the described technique.

Main Methods:

  • A gradient separation technique was employed, followed by isotonic ammonium chloride haemolysis.

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  • The technique was compared with two other established methods for blood cell separation.
  • The purity of isolated cells was assessed for the absence of lymphocytes and erythrocytes.
  • The functional capacity of isolated PMNs was measured by their ability to generate reactive oxygen species.

    • Main Results:

    • The gradient separation technique followed by haemolysis yielded a high number of polymorphonuclear neutrophils (PMNs).
    • This method achieved excellent purity, with no detectable lymphocyte or erythrocyte contamination.
    • Over 50 x 10^6 human PMNs were isolated from a 15 ml blood sample.
    • The isolated PMNs exhibited a significant capacity for reactive oxygen species generation.

    Conclusions:

    • The gradient separation technique combined with isotonic ammonium chloride haemolysis is a highly effective method for isolating pure polymorphonuclear neutrophils (PMNs).
    • This method provides a high yield of functional PMNs suitable for in vitro studies.
    • The technique offers a reliable approach for researchers requiring pure PMNs for immunological and cellular assays.