Isolation of Mouse Neutrophils.
Isolation of Mouse Neutrophils.
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DOI：
10.1002/0471142735.im0320s110
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发表时间：
2015-08-03
影响因子：
--
通讯作者：
Lionakis, Michail S
中科院分区：
文献类型：
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作者：
Swamydas, Muthulekha;Luo, Yi;Lionakis, Michail S
中性粒细胞代表对抗细菌和真菌病原体的第一道防线。事实上，患有遗传性和后天性中性粒细胞定性和定量缺陷的患者发生细菌和真菌感染并遭受这些感染的不良后果的风险很高。因此，旨在确定稳态条件下和感染期间调节中性粒细胞效应功能的分子因素的研究对于制定增强中性粒细胞功能和改善感染个体的结果的策略至关重要。本单元描述了一种基于密度梯度离心的可重复的方案，可应用于任何实验室，从稳定状态下和白色念珠菌感染后的小鼠骨髓中收获大量高度富集和高度存活的中性粒细胞，如 UNIT 中所述。在另一个协议中，我们还提出了一种将温和的酶组织消化与阳性免疫磁选技术或荧光激活细胞分选（FACS）相结合的方法，直接从小鼠组织（如肾脏、肝脏或脾脏）中收获高纯度和高活性的中性粒细胞制剂。最后，包括从小鼠腹腔液和外周血中分离中性粒细胞的方法。通过这些方案分离的小鼠中性粒细胞可用于检查离体细胞功能的几个方面，包括病原体结合、吞噬作用和杀伤、中性粒细胞趋化性、氧化爆发、脱粒和细胞因子产生，以及用于进行中性粒细胞过继转移实验。
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.