Department of Biological Sciences, University of Massachusetts Lowell, Lowell, MA, USA.
Methods Mol Biol. 2020;2087:93-106. doi: 10.1007/978-1-0716-0154-9_7.
Two critical functional responses of neutrophils are chemotaxis, a response driven by concentration gradients of chemokines released by infected or inflamed tissues, and production of reactive oxygen species (ROS), molecules essential to their capacity to kill pathogens. Assays to accurately test each response have been important to assess efficacies of pharmaceuticals predicted to block recruitment of neutrophils or attenuate their ROS production. Identified antagonists to neutrophil functions may help to reduce tissue damage following inflammation. Described are detailed assays to test these functions, along with steps to generate neutrophils from ex vivo-cultured murine bone marrow that produce robust responses in either assay. The first function protocol details a quantitative assay for chemotaxis that involves culture plates with dual chamber wells that separate cells from a chemokine with small pore-sized membranes. Quantitative measurements of cell numbers in the chemokine-containing chamber are performed with either fluorescence or luminescence detection reagents, which provide signals directly proportional to the numbers of migrated cells. Multiwell plates are used for rapidly testing a variety of conditions and/or chemoattractants. Described in the second function protocol is an assay to measure ROS produced by stimulated neutrophils, again using a multiwell platform for rapid, quantitative measurements of several conditions simultaneously.
中性粒细胞的两个关键功能反应是趋化作用，这是一种由感染或炎症组织释放的趋化因子浓度梯度驱动的反应，以及活性氧物质（ROS）的产生，这些物质对于其杀死病原体的能力至关重要。准确测试每种反应的测定方法对于评估预计阻止中性粒细胞募集或减弱其 ROS 产生的药物的疗效非常重要。鉴定中性粒细胞功能的拮抗剂可能有助于减少炎症后组织损伤。本文描述了详细的测定方法来测试这些功能，以及从体外培养的鼠骨髓中产生产生强烈反应的中性粒细胞的步骤，这些反应在任一种测定方法中都可以进行。第一个功能方案详细说明了一种用于趋化作用的定量测定方法，该方法涉及具有双室井的培养板，该培养板通过小孔径的膜将细胞与趋化因子分离。用荧光或发光检测试剂对趋化因子腔中细胞数量进行定量测量，这些试剂提供的信号与迁移细胞的数量直接成正比。多微孔板用于快速测试各种条件和/或趋化剂。第二个功能方案中描述了一种用于测量刺激中性粒细胞产生的 ROS 的测定方法，同样使用多微孔板平台同时快速定量测量几种条件下的 ROS 产生情况。
