The aim of the following experiment is to determine the role of polarized macrophages in a biological process in vivo. This is achieved by first depleting macrophages from mice. Next, new macrophages, which express unique features and have distinct functions are generated.
Then the polarized macrophages are adoptively transferred into mice to determine their role in the biological process of interest. Ultimately, differences in macrophage mediated inflammation based on clinical and histological disease can be measured. The main advantage of this technique over existing methods like crossing mice onto a CD 11 BDTR background is that this method is faster than breeding mice.
For experiments. It's relatively easy and cost-effective and can be performed with mice of any genetic background. Though this method can provide insight into the role of macrophages in the intestine, it can also be applied to a variety of other organs, including liver, spleen, and lung.
Two hours prior to injection remove tubes of liposomes and PBS from four degrees Celsius storage. When the liposomes have reached room temperature, invert the tubes eight to 10 times to ensure an even distribution. Then load 200 microliters of the warm to liposome suspension into a one milliliter syringe and attach a 26 gauge needle.
Roll the syringe between the palms of both hands and invert it six times to distribute the liposome solution evenly prior to injection. Now use one hand to scruff, a mouse just below the ears, holding enough skin to immobilize its head and limbs, and then tilt the mouse so its head is slightly towards the ground. Then holding the syringe at a 30 to 40 degree angle.
Insert the needle into the lower right quadrant of the abdomen and inject 200 microliters of liposome solution. After euthanizing an eight to 12 week old mouse and fastening the limbs in a stretched out position, remove the femurs and tibias cutting away as much muscle as possible during the dissection process. Then load a five milliliter syringe with IMDM with 10%FCS.
Attach a 26 gauge needle and flush the marrow from the bones. Dilute the bone marrow aspirates in 40 milliliters of IMDM with FCS, and then transfer the cells to a 75 centimeter square Tissue culture flask. Incubate the culture at 37 degrees Celsius and 5%carbon dioxide after four hours.
Collect the supinate in a 50 milliliter conical tube and centrifuge the non-adherent progenitors for five minutes at 300 times G and room temperature. After counting, culture the recovered cells at a 0.5 times 10 to the six cells per milliliter concentration in complete medium in fresh 75 centimeter square flasks. On day four, spin down the non-adherence cells and return them to the flasks in fresh media.
On day seven, discard the non-adherence cells, replacing them with fresh media. On day 10, discard the media. Then add five milliliters of cell dissociation buffer to the flask.
After five minutes, firmly bang the side of the flask with the heel of the palm of one hand several times. Examine the cells under a microscope to ensure that they have lifted off the bottom of the flask. Then pipette the resuspended cells into a 15 milliliter conical tube.
Wash the flask with an additional 10 milliliters of IMDM plus FCS, and then spin down the cells for five minutes at 300 times G and room temperature. After counting, resuspend the recovered cells at a concentration of 10 to the seven cells per milliliter in sterile PBS reserving one times 10 to the six macrophages for macrophage phenotype assessments begin by equipping a one milliliter syringe filled with 100 microliters at the just prepared macrophage suspension with a 26 gauge needle. Next warmer mouse using a heat lamp for five to 10 minutes to dilate the tail vein.
After transferring the mouse to a restraining device, rotate the tail 90 degrees so the vein is facing upward. Clean the injection site with an alcohol swab and then holding the needle with the bevel side facing up. And at a slight angle, insert the needle almost parallel to the vein.
If the needle is inserted properly, no resistance should be felt and the vein should become clear after injection following the injection, apply gentle pressure to the injection site until the bleeding stops. These immunohistochemical sections of a mouse colon demonstrate that intraperitoneal injection of clodronate containing liposomes results in a decrease in F four 80 positive macrophages during DSS induced colitis as evidenced by the decrease in F four 80 staining in brown as compared to control PBS injected mice arginase one positive. Alternatively, activated macrophages in brown are also successfully depleted using clodronate liposome injections.
The blue stain in this and the previous figure represents nuclei as the bar graph show quantitative assessment of F four 80 positive MCSF derived macrophages and arginase one positive. Alternatively, activated MCSF derived plus IL four treated macrophages indicates that both populations are significantly depleted in mice that receive intraperitoneal injections of clodronate liposomes. These data demonstrate the results from a typical grease assay, which measures the levels of nitric oxide in response to lipopolysaccharide by detecting its downstream metabolite nitrite.
Thus classically activated MCSF derived macrophages produce significantly higher levels of nitrite compared to alternatively activated MCSF derived plus IL four treated macrophages. Classically activated macrophages also produce a higher IL 12 P 70 to IL 10 ratio compared to alternatively activated macrophages as measured by ELA consistent with an alternatively activated phenotype. MCSF derived macrophages treated with IL four constitutively express YM one and arginase one as detected by western immuno flossing.
Here, the total number of classically activated M1 macrophages and the number of alternatively activated M two macrophages in enumerated in histological sections from mice injected with polarized bone marrow derived macrophages are shown. The C data bar indicates control mice that received a PBS injection without macrophages. The impact that the transfer of polarized macrophages has on the colon during DSS induced colitis is indicated by the disease activity index, an additive score based on weight loss, rectal bleeding, and decreased stool consistency.
Note that M1 macrophages do not impact inflammation, whereas M two macrophages significantly reduce the disease activity index. Therefore, these results demonstrate the dex vivo derived adoptively transferred macrophages can traffic to the colon and impact induced inflammation After its development. This technique paved the way for researchers studying macrophage biology to explore the role of macrophage polarization in mice.
After watching this video, you should have a good understanding of how to determine the role of polarized macrophages in homeostasis and in disease in mice.
