A Simple, Reproducible, Inexpensive, Yet Old-Fashioned Method for Determining Phagocytic and Bactericidal Activities of Macrophages

Abstract

Macrophages (Mφ) play a pivotal role in the protection system by recognizing and eliminating invading pathogenic bacteria. Phagocytosis and the killing of invading bacteria are major effector functions of Mφ. Although the phagocytic and bactericidal activities of Mφ have been analyzed via several methods using a light microscope, a fluorescence microscope, or a fluorescence-activated cell sorter, expensive materials and equipment are usually required, and the methods are rather complicated. Moreover, it is impossible to determine both the phagocytic and bactericidal activities of Mφ simultaneously using these methods. In this review, we describe a simple, reproducible, inexpensive, yet old-fashioned method (antibiotic protection assay) for determining the phagocytic and bactericidal activities of Mφ.

Keywords: Anti-bacterial agent; gentamicin; macrophage; phagocytosis.

Fig. 1. An antibiotic protection assay. Two sets of Mϕ (one for the determination of phagocytic activity and the other for the determination of bactericidal activity) are incubated for a short period of time with bacteria in CM to be engulfed by Mϕ. To determine phagocytic activity, Mϕ are washed with CM containing antibiotic to kill extracellular bacteria followed by CM. They are then treated with saponin to release bacteria from Mϕ, and CFUs are determined [CFU (P)]. To determine bactericidal activity, Mϕ infected with bacteria are further incubated for a short period of time in CM containing antibiotic to kill intracellular bacteria followed by CM. They are then treated with saponin, and the number of viable bacteria in Mϕ is determined by counting the CFU after washing with CM [CFU (B)]. Bactericidal activity of Mϕ can be calculated by comparing CFU (P) with CFU (B). CM, complete medium; CFU, colony-forming units; Mϕ, macrophages.

Fig. 2. Influence of antibiotics with high and low molecular weight on bacteria in Mϕ. Mϕ engulf bacteria by forming specific vacuoles called phagosomes. An antibiotic with a low molecular weight penetrates into the cytosol and kills both extracellular and intracellular bacteria (A). An antibiotic with a high molecular weight is unable to penetrate into the cytosol and thus kills only extracellular bacteria (B). Mϕ, macrophages.

Fig. 3. Killing mechanism of GM. GM inhibits translation of mRNA by binding to the 30S subunit of the ribosome. The irreversible binding of GM to the ribosome causes the misreading of the codons, which in turn causes an error in the proofreading process of translation, leading to incorrect protein expression and bacterial cell death. GM, gentamicin.

Fig. 4. Experimental procedure for determining the phagocytic and bactericidal activities of Mϕ using a GM protection assay. Two tissue culture plates are prepared; one to determine phagocytic activity and the other to determine bactericidal activity. Mϕ are incubated in CM for 120 min to adhere to the bottom of tissue culture plates and then incubated with bacteria (Mϕ:bacteria=1:10) for a short period of time in CM to ingest bacteria. To remove non-ingested bacteria, cells are washed three times with CM containing the optimal concentration of GM followed by CM, and CFUs are then determined [CFU (P)]. Infected Mϕ incubated in another plate are further incubated for a short period of time in CM containing the optimal concentration of GM. During this period, engulfed bacteria are killed by Mϕ. Cells are washed three times with CM, and the number of viable bacteria in Mϕ can also be determined by counting CFU after washing with CM [CFU (B)]. Bactericidal activity of Mϕ can be calculated by comparing CFU (P) with CFU (B) after saponin treatment and sonication. GM, gentamicin; CM, complete medium; CFU, colony-forming units; Mϕ, macrophages.

Fig. 5. Influence of GM on Mϕ. (A) RAW264 were incubated with CM containing various concentrations of GM for 48 h, and the morphological changes were observed under phase contrast microscope. Representative data from two independent experiments are shown. (B) RAW264 were incubated with CM containing various concentrations of GM for 48 h, and their viabilities were enumerated via trypan blue exclusion test. Data are presented as mean±SD of two independent experiments. ^*p<0.05: 0 vs. 100 or 500. N.D., not detectable; GM, gentamicin; CM, complete medium; Mϕ, macrophages.

Fig. 6. Effects of saponin and sonication on plasma and phagosomal membranes of Mϕ. Saponin interacts with cholesterols residing in plasma and phagosomal membranes and forms pores in lipid bilayers. After sonication, internalized bacteria are released. Mϕ, macrophages.