Measurement of Bacterial Ingestion and Killing by Macrophages

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.

GDF3 Protects Mice against Sepsis-Induced Cardiac Dysfunction and Mortality by Suppression of Macrophage Pro-Inflammatory Phenotype

“…Emerging evidence has shown that macrophage phenotypic alterations play a pivotal role during sepsis [5]. In general, M1 macrophages are pro-inflammatory; their apoptosis during the process of pathogen clearance simultaneously dampens the inflammatory response and transition of M1 to tissue-repairing M2 macrophages [7,24]. Therefore, understanding the mechanisms that control macrophage polarization would be crucial for the improvement of immune therapy against sepsis.…”

“…For example, M1 macrophages are antigen-presenting cells that can be induced by LPS or IFN-γ. They are mainly responsible for producing pro-inflammatory mediators like tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), interleukin 1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1), and iNOS [6,7]. During early phase of sepsis, numbers of macrophages with M1 phenotype rise significant with primary function of killing bacteria [7].…”

“…They are mainly responsible for producing pro-inflammatory mediators like tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), interleukin 1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1), and iNOS [6,7]. During early phase of sepsis, numbers of macrophages with M1 phenotype rise significant with primary function of killing bacteria [7]. On the flip side, M2 macrophages could be induced by IL-4, they are recognized by anti-inflammatory and tissue-repairing characteristics at later stage of sepsis [6].…”

Live cell imaging to understand monocyte, macrophage, and dendritic cell function in atherosclerosis

“…Cells that have been plated on cover glass-bottom dishes or cytospun can be analyzed for shape to help determine their phenotype, though this can also be performed on an imaging flow cytometer ( Majka et al, 2012 ). Sorted cells can also be used for in vitro functional assays like antigen presentation ( Choi et al, 2009 ), cytokine production by cytometric bead array ( Morgan et al, 2004 ), phagocytosis ( Xu et al, 2010 ), efferocytosis of apoptotic cells ( Friggeri et al, 2010 ), and killing of pathogens ( Drevets et al, 2015 ).…”

A primitive type of renin-expressing lymphocyte protects the organism against infections

“…These bacteria were used for SEM (below). For bacterial ingestion and killing assays, we used Salmonella typhimurium (ATCC 14028) and modified the protocol as described (25,26). Briefly, we isolated peritoneal cells from WT or renin KO mice.…”