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Updated: Jul 16, 2025
Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments
Published on: July 10, 2013
Isolation of Mouse Neutrophils
Andrew L Wishart1, Muthulekha Swamydas1, Michail S Lionakis1
1Fungal Pathogenesis Section, Laboratory of Clinical Immunology & Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Abstract:
Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited or acquired qualitative and quantitative neutrophil defects are at high risk for developing bacterial and fungal infections and suffering adverse outcomes from these infections. Therefore, research aiming at defining the molecular factors that modulate neutrophil effector function under homeostatic conditions and during infection is essential for devising strategies to augment neutrophil function and improve the outcomes of infected individuals. This article describes reproducible density-gradient-centrifugation-based as well as positive and negative immunomagnetic selection protocols that can be applied in any laboratory to harvest large numbers of highly enriched and highly viable neutrophils from the bone marrow of mice. In another protocol, we also present a method that combines gentle enzymatic tissue digestion with a positive immunomagnetic selection technique or fluorescence-activated cell sorting (FACS) to harvest highly pure and highly viable preparations of neutrophils directly from mouse tissues such as the kidney, the liver, or the spleen. Mouse neutrophils isolated by these protocols can be used to examine several aspects of cellular function ex vivo, including pathogen binding, phagocytosis, and killing, neutrophil chemotaxis, oxidative burst, degranulation, and cytokine production, and for performing neutrophil adoptive transfer experiments. © 2023 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol 1: Isolation of Neutrophils from Mouse Bone Marrow Using Positive Immunomagnetic Separation Alternate Protocol 1: Purification of Neutrophils from Bone Marrow Using Negative Immunomagnetic Separation Alternate Protocol 2: Purification of Neutrophils from Bone Marrow Using Histopaque-Based Density Gradient Centrifugation Basic Protocol 2: Isolation of Neutrophils from Mouse Tissues Using Positive Immunomagnetic Separation Alternate Protocol 3: Isolation of Neutrophils from Mouse Tissues Using FACS.
Insights
This study provides standardized protocols for isolating neutrophils, crucial immune cells, from mouse bone marrow and tissues. These methods ensure high purity and viability for studying neutrophil function in infection and immunity.
Area of Science:
- Immunology
- Cell Biology
- Hematology
Background:
- Neutrophils are vital for innate immunity against bacterial and fungal infections.
- Defects in neutrophil number or function increase susceptibility to severe infections.
- Understanding neutrophil function is key to developing treatments for infectious diseases.
Purpose of the Study:
- To describe reproducible methods for isolating high-purity, viable neutrophils from mouse bone marrow and tissues.
- To provide standardized protocols applicable in diverse laboratory settings.
- To enable detailed ex vivo analysis of neutrophil functions and adoptive transfer experiments.
Main Methods:
- Density-gradient centrifugation for neutrophil isolation from bone marrow.
- Positive and negative immunomagnetic selection for bone marrow neutrophil purification.
- Enzymatic tissue digestion combined with immunomagnetic selection or FACS for tissue neutrophil isolation.
Main Results:
- Established protocols yield large numbers of highly enriched and viable neutrophils.
- Methods are effective for isolating neutrophils from mouse bone marrow, spleen, liver, and kidney.
- Isolated neutrophils are suitable for functional assays including phagocytosis, oxidative burst, and cytokine production.
Conclusions:
- These validated protocols facilitate robust research into neutrophil biology and function.
- Standardized isolation techniques are essential for reproducible studies in immunology and infectious disease.
- The methods support investigations into neutrophil-mediated immunity and the development of novel therapeutic strategies.
