Related Experiment Videos
Method Matters: Effect of Purification Technology on Neutrophil Phenotype and Function
Marfa Blanter1, Seppe Cambier1, Mirre De Bondt1
1Laboratory of Molecular Immunology, Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven, Leuven, Belgium.
Frontiers in Immunology
|February 28, 2022
Summary
Neutrophil isolation methods impact cell function. Density-gradient purification artificially activates neutrophils, while immunomagnetic isolation yields quiescent cells more responsive to stimuli, affecting experimental outcomes.
Area of Science:
- Immunology
- Cell Biology
- Hematology
Background:
- Neutrophils are crucial for innate immunity, responding rapidly to infection and injury.
- Their short lifespan and inability to be cultured ex vivo necessitate immediate purification from fresh blood.
- Existing purification methods may alter neutrophil phenotype and function, impacting experimental reliability.
Purpose of the Study:
- To compare the effects of density-gradient purification versus immunomagnetic isolation on neutrophil phenotype and function.
- To determine how these isolation methods influence neutrophil responses to inflammatory stimuli.
- To provide guidance on selecting appropriate neutrophil isolation techniques for specific experimental assays.
Main Methods:
- Comparison of density-gradient and immunomagnetic neutrophil isolation techniques.
- Assessment of neutrophil phenotype and function, including polarization, reactive oxygen species (ROS) production, neutrophil extracellular trap (NET) release, degranulation, and phagocytosis.
- Chemotaxis assays using Multiscreen, under-agarose, and Boyden methods.
- Flow cytometry analysis of cell surface marker expression (e.g., CD66b, FPR1, CD35, CXCR3, MHC-II, CD14).
Main Results:
- Density-gradient neutrophils exhibited higher spontaneous ROS release, NETosis, and degranulation, indicating artificial activation.
- Immunomagnetic neutrophils were more responsive to inflammatory stimuli in polarization, ROS production, phagocytosis, NETosis, and degranulation assays.
- No significant difference in chemotaxis was observed in Multiscreen and under-agarose assays, but Boyden assays showed reduced chemotaxis in immunomagnetic neutrophils.
- Density-gradient purification led to increased expression of activation markers (CD66b, FPR1, CD35) and atypical surface molecules (CXCR3, MHC-II, CD14).
Conclusions:
- Density-gradient purification induces artificial neutrophil activation, altering their baseline characteristics.
- Immunomagnetic isolation yields less activated neutrophils that are more responsive to inflammatory stimuli, making it suitable for studying neutrophil functions like polarization, phagocytosis, ROS production, degranulation, and NETosis.
- Density-gradient purification remains more appropriate for Boyden chemotaxis assays.
- Choosing the correct isolation method is critical for accurate neutrophil research.