UNIT 14.6 Measurement of Bacterial Ingestion and Killing by Macrophages. (1st April 2015)

• Record Type: Journal Article
• Authors: Drevets, Douglas A.; Canono, Beth P.; Campbell, Priscilla A.
• Is Part Of: Current protocols in immunology. Volume 109(2015)
• Page Start: 14.6.1
• Page End: 14.6.17
• Publication Date: 2015-04-01
• Subjects: macrophages -- internalization -- ingestion of bacteria -- phagocytosis -- bacterial killing; Immunology -- Laboratory manuals; Medical protocols; Immunologic Techniques; Medical protocols; Immunology; Laboratory manuals; Reference works; 571.96028
• DOI: 10.1002/0471142735.im1406s109
• Languages: English
• ISSNs: 1934-3671

Abstract

Abstract : 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. © 2015 by John Wiley & Sons, Inc.