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Related Experiment Videos

Labeled polymorphonuclear leukocytes: a comparison of methodology.

S Chowdhury1, M L Brown, M K Dewanjee

  • 1Mayo Clinic and Foundation, Rochester, MN 55905.

International Journal of Radiation Applications and Instrumentation. Part B, Nuclear Medicine and Biology
|January 1, 1988
PubMed
Summary

Volex sedimentation with hypotonic lysis (VSHL) offers a viable method for isolating pure polymorphonuclear neutrophils. This technique provides comparable cell viability and function to Ficoll-Hypaque separation, making it a practical alternative for granulocyte isolation.

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Area of Science:

  • Immunology
  • Cell Biology
  • Hematology

Background:

  • Accurate isolation of polymorphonuclear neutrophils (PMNs) is crucial for immunological studies.
  • Existing methods like Ficoll-Hypaque (FH) separation are effective but can be time-consuming or costly.
  • Alternative techniques for PMN isolation require thorough evaluation for purity, viability, and functional integrity.

Purpose of the Study:

  • To compare the efficacy of volex sedimentation (VS), volex sedimentation with hypotonic lysis (VSHL), and Ficoll-Hypaque (FH) gradient separation for isolating PMNs.
  • To assess the purity, viability, and functional capacity (chemotaxis, survival) of PMNs obtained by each method.
  • To determine if VSHL is a suitable alternative to FH for obtaining pure granulocyte preparations.

Main Methods:

Related Experiment Videos

  • Separation of PMNs from human and canine whole blood using VS, VSHL, and FH techniques.
  • Labeling of isolated cells with 111In-oxine and 111In-tropolone for survival studies.
  • Assessment of cell viability using the Trypan blue exclusion test.
  • Evaluation of granulocyte chemotactic response to E. coli in agarose plates.
  • Determination of 111In-labeled granulocyte survival in a canine model.

    • Main Results:

    • All three isolation methods (VS, VSHL, FH) resulted in 100% cell viability.
    • Chemotactic migration and leukocyte survival times were comparable across all tested techniques.
    • The VSHL technique significantly reduced the contamination of red blood cells and platelets compared to VS, approaching the purity of FH separation.
    • PMN preparations using VSHL demonstrated fewer contaminating red blood cells and platelets.

    Conclusions:

    • VSHL is an effective method for isolating polymorphonuclear neutrophils with high purity.
    • VSHL provides comparable cell viability and functional characteristics to Ficoll-Hypaque separation.
    • VSHL represents a practical and acceptable alternative to Ficoll-Hypaque for obtaining relatively pure granulocyte preparations.