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Related Experiment Video

Updated: Jul 8, 2025

Isolation of Human Neutrophils from Whole Blood and Buffy Coats
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Isolation methods determine human neutrophil responses after stimulation.

Vanessa Krémer1,2, Ophélie Godon1, Pierre Bruhns1

  • 1Institut Pasteur, Université Paris-Cité, Institut national de la santé et de la recherche (INSERM) Unité mixte de recherche (UMR)1222, Antibodies in Therapy and Pathology, Paris, France.

Frontiers in Immunology
|December 11, 2023
PubMed
Summary

Choosing the right neutrophil isolation method is crucial. Methods without red blood cell (RBC) lysis, like negative immunomagnetic selection, yield less activated and more responsive neutrophils for accurate study.

Keywords:
functional analysishuman bloodisolationneutrophil (PMN)phenotype

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Last Updated: Jul 8, 2025

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Area of Science:

  • Immunology
  • Cell Biology
  • Hematology

Background:

  • Neutrophils are critical immune cells but challenging to study due to short lifespan, lack of proliferation, and genetic manipulation resistance.
  • Neutrophil activation during blood collection and processing can confound experimental results, impacting their purity, activation status, and responsiveness.

Purpose of the Study:

  • To evaluate the impact of five common neutrophil isolation methods on neutrophil yield, purity, activation, and responsiveness.
  • To identify isolation protocols that best preserve neutrophil integrity and function for downstream analysis.

Main Methods:

  • Assessed five widely used neutrophil isolation techniques.
  • Monitored surface markers, reactive oxygen species (ROS) production, and DNA release (as a surrogate for neutrophil extracellular trap [NET] formation).
  • Compared outcomes from density gradient centrifugation with and without red blood cell (RBC) lysis against negative immunomagnetic selection.

Main Results:

  • Neutrophils isolated using negative immunomagnetic selection and density gradient methods without RBC lysis showed minimal activation, closely resembling neutrophils in whole blood.
  • These methods resulted in less activated and more responsive neutrophils compared to density gradient methods requiring RBC lysis.
  • Isolation protocols significantly influenced neutrophil activation status and functional responses.

Conclusions:

  • The choice of neutrophil isolation method critically impacts experimental outcomes, affecting cell activation and responsiveness.
  • Negative immunomagnetic selection and density gradient methods without RBC lysis are recommended for preserving neutrophil integrity.
  • Standardization of neutrophil isolation protocols is essential for reliable neutrophil subset characterization and reproducible research.