Fungal Pathogenesis Section, Laboratory of Clinical Immunology & Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Curr Protoc. 2023 Sep;3(9):e879. doi: 10.1002/cpz1.879.
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.
中性粒细胞是抵御细菌和真菌病原体的第一道防线。事实上，患有遗传性或获得性中性粒细胞质量和数量缺陷的患者发生细菌和真菌感染的风险很高，并且会因这些感染而产生不良后果。因此，研究旨在确定调节中性粒细胞效应功能的分子因素，对于制定增强中性粒细胞功能和改善感染个体预后的策略至关重要。本文描述了基于密度梯度离心的可重现性以及正选和负选免疫磁珠分离方案，这些方案可应用于任何实验室，从小鼠骨髓中收获大量高度富集和高活力的中性粒细胞。在另一个方案中，我们还介绍了一种将温和的酶组织消化与正选免疫磁珠分离技术或荧光激活细胞分选（FACS）相结合的方法，从肾脏、肝脏或脾脏等小鼠组织中直接收获高度纯净和高活力的中性粒细胞制剂。通过这些方案分离的小鼠中性粒细胞可用于体外研究细胞功能的多个方面，包括病原体结合、吞噬作用和杀伤作用、中性粒细胞趋化性、氧化爆发、脱颗粒作用和细胞因子产生，以及进行中性粒细胞过继转移实验。 © 2023 Wiley Periodicals LLC。本文的贡献者是美国政府雇员，其工作在美国属于公有领域。 基本方案 1：使用正选免疫磁珠分离法从鼠骨髓中分离中性粒细胞 可选方案 1：使用负选免疫磁珠分离法从骨髓中纯化中性粒细胞 可选方案 2：使用基于 Histopaque 的密度梯度离心法从骨髓中纯化中性粒细胞 基本方案 2：使用正选免疫磁珠分离法从鼠组织中分离中性粒细胞 可选方案 3：使用 FACS 从鼠组织中分离中性粒细胞
