Isolation of Mouse Neutrophils

Abstract

Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited and 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 outcome of infected individuals. This unit describes a reproducible density gradient centrifugation-based protocol that can be applied in any laboratory to harvest large numbers of highly enriched and highly viable neutrophils from the bone marrow of mice both at the steady state and following infection with Candida albicans as described in UNIT. 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. Finally, methods for isolating neutrophils from mouse peritoneal fluid and peripheral blood are included. Mouse neutrophils isolated by these protocols can be used for examining 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.

Keywords: density gradient centrifugation; isolation; magnetic separation; mouse bone marrow; mouse tissue; neutrophils; sorting.

Figure 3.20.1

Separation of casein-induced peritoneal neutrophils following purification by Percoll density gradient centrifugation. The faint upper band contains primarily lymphocytes and macrophages (L,M), the broad middle band includes neutrophils and other granulocytes such as eosinophils (polymorphonuclear leukocytes; PMN), and the bottom band includes erythrocytes (E).

Figure 3.20.2

Purity (left panel) and viability (right panel) of neutrophils isolated from the bone marrow of an uninfected C57BL/6 mouse using the Histopaque-based gradient centrifugation method.

Figure 3.20.3

Purity (left panel) and viability (right panel) of neutrophils isolated from the kidneys of a Candida albicans-infected C57BL/6 mouse using the Ly6G-based positive immunomagnetic selection method.