Measurement of bacterial ingestion and killing by macrophages.
Measurement of bacterial ingestion and killing by macrophages.
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DOI：
10.1002/0471142735.im1406s109
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发表时间：
2015-04-01
影响因子：
--
通讯作者：
Campbell, Priscilla A
中科院分区：
文献类型：
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作者：
Drevets, Douglas A;Canono, Beth P;Campbell, Priscilla A
本单元介绍了允许准确测量巨噬细胞吞噬和杀死细菌的测定。第一个基本协议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需要单独的结合和内化步骤，本文描述的测定也将确定结合至巨噬细胞的细菌实际上被内化的程度。在替代方案中描述了两种有效的方法。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。该单位还提出了两个协议，以衡量巨噬细胞杀死细菌的能力，它已经内化。第二个基础是一个简单的测定，其中细菌菌落计数之前和之后的一个杀灭期。通过琼脂平板上CFU细菌减少证明了杀菌活性。由于去除残留的细胞外有机体至关重要，该方案提出了两个替代步骤来实现这一点：洗涤程序和更严格的方法，其中细胞通过蔗糖沉淀。另一种方案描述了一种基于细菌代谢来测量细菌活力的方法，其中通过比色法测量吸光度来监测细菌辅酶Ⅱ酶介导四唑盐还原为紫色甲瓒的能力。
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.