Phagocytosis Assay

Phagocytosis is one of the first lines of defense against invading microorganisms. Macrophages play a central role in this process as a host defends against intruding foreign bodies. This phagocytosis assay was developed in order to further elucidate the level at which macrophage function is compromised, if at all, in the presence of cannabinoids.

3.4.1. Macrophage Treatment With Foreign Antigen

1. Thioglycolate-elicited peritoneal macrophages from CB2R+/+ or CB2R-/- are plated and treated with cannabinoids as indicated in Subheading 3.1.3. After incubating with cannabinoids for 1 h, in order to reduce cannabinoid interference, media is removed and 1 mL of fresh complete media is added prior to the addition of the foreign particles. Ten to 25 |L of opsonized microspheres or 100:1 bacteria to macrophage are added and allowed to incubate for 1 h at 37°C.

2. Following the 1-h incubation, decant the complete medium to remove non-phago-cytized particles. Phagocytosis is stopped by applying several rinses of 0.5-mL cold PBS to each well.

3. Next, fix macrophages onto the coverslips with 0.25 mL warm fixing medium and incubate for 15 min at 55°C. Rinse cells several times with 0.5 mL warm PBS.

4. Permeabilize macrophages with 0.25 mL of warm permeabilization medium for 10 min at 37°C. Wash cells two times with 0.5 mL of warm PBS, with a 5-min incubation step between washes, to ensure complete elimination of the detergent, Triton X-100. Do a final wash with 0.25 mL of sterile distilled water in order to remove all the salts prior to mounting.

5. To mount the cover slips, one drop of warm mounting medium is dropped onto a clean slide. Immediately pry the cover slip from the bottom of the well using an 18-gage needle and sterile tweezers. Flip the cover slip so that the adherent macrophages are preserved and mounted directly on top of the mounting medium.

E3 control ^ 10nM WIN

cell type

Fig. 3. WIN55-212,2 enhances CB2R+/+ T cell IL-2 secretion, but not CB2R-/- T cell IL-2 secretion. IL-2 secretion was determined using the IL-2 kit from BD Biosciences as indicated in Subheading 3. Cells were plated at 2 x 105 cells/0.1 mL/well in complete media. Cells were treated with 2.5 |ig/mL ConA and the indicated A9-THC concentration for 72 h. Data are expressed as the mean of quadruplicate samples + standard deviation. Vehicle is absolute ethanol.

cell type

Fig. 3. WIN55-212,2 enhances CB2R+/+ T cell IL-2 secretion, but not CB2R-/- T cell IL-2 secretion. IL-2 secretion was determined using the IL-2 kit from BD Biosciences as indicated in Subheading 3. Cells were plated at 2 x 105 cells/0.1 mL/well in complete media. Cells were treated with 2.5 |ig/mL ConA and the indicated A9-THC concentration for 72 h. Data are expressed as the mean of quadruplicate samples + standard deviation. Vehicle is absolute ethanol.

Finally, secure the cover slips with nail polish and allowed to dry overnight prior to viewing under the microscope.

3.4.2. Microscopic Analysis

1. The red-dyed latex beads are viewed with light microscopy under 40X magnification. The percent phagocytosis is determined by comparing the number of phago-cytosing macrophages vs total macrophages present in the field. Macrophages with 5 or more red-dyed micropheres are considered positive for phagocytosis. A total of 200 macrophages are counted per mount.

2. Phagocytosis of microspheres and bacteria is determined using phase-contrast microscopy with a green fluorescent filter. Fluorescent-dyed microspheres and

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Fig. 4. A9-THC inhibits phagocytosis by CB2R+/+ macrophages, but not of CB2R-/-macrophages. Thioglycolate-induced peritoneal macrophages were plated at a density of 0.5-1 x 106 cells/well as indicated in text and treated with the indicated concentrations of THC or 1% ethanol as vehicle control for 1 h. After 1 h, the cells were challenged with opsonized red-dyed microspheres. The cells were then processed for light microscopy as indicated in the text. Phagocytic activity, expressed at % phagocytosis, was determined by counting cells that had engulfed five or more red-dyed microspheres out of a field of 200 cells. Data are representative of three experiments and are expressed as the mean of triplicate samples + standard deviation.

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Fig. 4. A9-THC inhibits phagocytosis by CB2R+/+ macrophages, but not of CB2R-/-macrophages. Thioglycolate-induced peritoneal macrophages were plated at a density of 0.5-1 x 106 cells/well as indicated in text and treated with the indicated concentrations of THC or 1% ethanol as vehicle control for 1 h. After 1 h, the cells were challenged with opsonized red-dyed microspheres. The cells were then processed for light microscopy as indicated in the text. Phagocytic activity, expressed at % phagocytosis, was determined by counting cells that had engulfed five or more red-dyed microspheres out of a field of 200 cells. Data are representative of three experiments and are expressed as the mean of triplicate samples + standard deviation.

GFP-expressing E. coli can be viewed under 20X or 40X magnification using a Nikon Eclipse TE300 microscope.

3. A field of interest is first visualized and photographed with the fluorescent filter. The same field is then photographed with the fluorescent filter. Resulting photographs are digitally overlayed using Metamorph Imaging Software, version 6.2r2 (Universal Imaging Corporation). An acceptable threshold of fluorescence inside and outside of the cell is then established using Metamorph. Based on the established parameters, quantification of phagocytosis is possible.

4. As seen in Fig. 4, THC inhibits phagocytic activity of thioglycolate-induced peritoneal CB2R+/+ macrophages, but not of CB2R-/- macrophages, suggesting that THC is, at least partially, acting through the CB2R. Similar results are obtained when using WIN55-212-2 (data not shown).

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