Nuzzi Paul A, Lokuta Mary A, Huttenlocher Anna
Department of Molecular and Cellular Pharmacology, University of Wisconsin Medical School, Madison, WI, USA.
Methods Mol Biol. 2007;370:23-36. doi: 10.1007/978-1-59745-353-0_3.
Neutrophils are the initial responders to bacterial infection or other inflammatory stimuli and comprise a key component of the innate immune response. In addition to their unique morphology and antimicrobial activity, neutrophils are characterized by the ability to migrate rapidly up shallow gradients of attractants in vivo. The directed migration of neutrophils, referred to as chemotaxis, requires the temporal and spatial regulation of intracellular signaling pathways allowing the neutrophil to detect a gradient of attractant, polarize, and migrate rapidly toward the highest concentration of the chemoattractant. A challenge to understanding neutrophil chemotaxis is the inherent difficulty encountered when working with primary neutrophils, which are difficult to purify in the resting state, are not easily transfected, are terminally differentiated, and have a short life span after purification. Here we discuss neutrophil purification methods and chemotaxis assays and provide methodology for working with a neutrophil-like cell line, the HL-60 promyelocytic leukemia cell line. We also discuss methods for HL-60 transfection using retroviral approaches and chemotaxis assays used with differentiated HL-60 cells.
中性粒细胞是对细菌感染或其他炎症刺激的初始应答者，是固有免疫反应的关键组成部分。除了其独特的形态和抗菌活性外，中性粒细胞的特征还在于能够在体内沿趋化因子的浅梯度快速迁移。中性粒细胞的定向迁移，即趋化作用，需要对细胞内信号通路进行时空调节，使中性粒细胞能够检测到趋化因子梯度、极化并迅速向趋化因子浓度最高的方向迁移。理解中性粒细胞趋化作用面临的一个挑战是在处理原代中性粒细胞时遇到的固有困难，原代中性粒细胞在静息状态下难以纯化，不易转染，是终末分化的，并且在纯化后寿命较短。在这里，我们讨论中性粒细胞的纯化方法和趋化分析，并提供使用中性粒细胞样细胞系HL-60早幼粒细胞白血病细胞系的方法。我们还讨论了使用逆转录病毒方法进行HL-60转染的方法以及用于分化的HL-60细胞的趋化分析。
