Drevets Douglas A, Canono Beth P, Campbell Priscilla A
Department of Medicine, University of Oklahoma Health Sciences Center and the Veterans Affairs Medical Center, Oklahoma City, Oklahoma.
Central Translational Research Center, National Jewish Health, Denver, Colorado.
Curr Protoc Immunol. 2015 Apr 1;109:14.6.1-14.6.17. doi: 10.1002/0471142735.im1406s109.
This unit presents assays that allow accurate measurement of phagocytosis and killing of bacteria by macrophages. The first basic protocol describes how to measure the ability of macrophages to ingest bacteria. Importantly, because macrophage phagocytosis entails separate binding and internalization steps, assays are described here that will also determine the extent to which bacteria bound to the macrophage are in fact internalized. Two effective methods to do this are described in alternate protocols. Both of these alternate protocols rely on enumeration of differentially labeled bacteria by fluorescence microscopy to distinguish intracellular from extracellular bacteria. The unit also presents two protocols to measure the ability of a macrophage to kill bacteria it has internalized. The second basic is a straightforward assay in which bacterial colonies are enumerated before and after a killing period. Bactericidal activity is evidenced by reduced CFU bacteria on agar plates. Because it is critical to remove residual extracellular organisms, the protocol presents two alternative steps to accomplish this: a washing procedure and a more stringent method in which cells are sedimented through sucrose. An alternate protocol describes a way to measure bacterial viability based on bacterial metabolism, in which the ability of bacterial dehydrogenases to mediate the reduction of a tetrazolium salt to purple formazan is monitored by measuring absorbance spectrophotometrically.
本单元介绍了一些检测方法，可用于准确测量巨噬细胞对细菌的吞噬作用和杀伤能力。第一个基本方案描述了如何测量巨噬细胞摄取细菌的能力。重要的是，由于巨噬细胞吞噬作用涉及单独的结合和内化步骤，因此这里介绍的检测方法还将确定与巨噬细胞结合的细菌实际上被内化的程度。替代方案中描述了两种有效的方法来实现这一点。这两种替代方案都依赖于通过荧光显微镜对差异标记的细菌进行计数，以区分细胞内细菌和细胞外细菌。本单元还介绍了两种检测巨噬细胞杀伤其内化细菌能力的方案。第二个基本方案是一种直接的检测方法，即在杀伤期前后对细菌菌落进行计数。琼脂平板上CFU细菌数量减少证明了杀菌活性。由于去除残留的细胞外生物体至关重要，该方案提出了两个替代步骤来实现这一点：一个洗涤程序和一个更严格的方法，即通过蔗糖使细胞沉淀。替代方案描述了一种基于细菌代谢来测量细菌活力的方法，其中通过分光光度法测量吸光度来监测细菌脱氢酶介导四唑盐还原为紫色甲臜的能力。
