The overall goal of these experiments is to determine the effects of antibody-based drugs on anti-inflammatory macrophage activation states using mouse bone marrow and peritoneal macrophages. This method can help answer key questions in the field of immunology, such as, do specific antibody drugs act by changing macrophages to an IL-10 producing anti-inflammatory activation state. The main advantage of this technique, is that it measures the response of primary mouse macrophages from the bone marrow in vitro and peritoneal cavity in vivo.
Perform this procedure in a sterile hood. Secure a euthanized mouse supine on a foam board with its limbs outstretched. Then, clean the legs with 70%ethanol.
Next, make a shallow cut about two millimeters deep to remove the skin and fur from the surface of each hind leg. Then, dissect the tissues to access the tibias and femurs. To remove the tibias and femurs, cut the bone and muscles just below the hip joint, and above the ankles.
Then, remove as much of the muscle tissue from the bones as possible. Next, clean the bones with 70%ethanol, and allow it to evaporate for one minute. Then, place the bones in a six well plate filled with bone flesh medium.
In the plate, trim one millimeter from either end of the femurs so that their inner cavities are exposed. Make certain a 26-gauge needle can access the cavities. Next, separate the tibia and femur from the knee joint.
Then, insert the needle attached to a 10 milliliter syringe into a bone cavity and collect the bone marrow. Pull the bone marrow from all four bones into a 50 milliliter conical tube with five milliliters of bone flesh medium. Pipe out the medium and marrow up and down several times.
Or vortex at a slow speed to gently disperse clumps in the suspension. Strings of marrow should be broken up into small flecks of marrow less than half a millimeter long. Next, top off the tube to 50 milliliters with bone flesh medium, and transfer it into a 75 square centimeter tissue culture flask.
Incubate the bone marrow for an hour. During the incubation, the matured cells will adhere to the flask, and the desired hematopoietic progenitors will remain in suspension. Transfer the suspension to a 50 milliliter conical tube and spin down the cells into a pellet.
Next, discard the supernatant and resuspend the cell pellet in five milliliters of MCSF culture medium. Then adjust the cell density to 500, 000 per milliliter and load 30 milliliters into a new 75 square centimeter flask. Continue culturing the cells, and on days four, seven, and 10, verify that the cells are adherent and slightly branched using an inverted phase contrast breakfield microscope.
When checking the cells, discard the medium containing the non-adherent cells. Then wash the adherent cells with IMDM one time, and add 30 milliliters of fresh MCSF culture medium back into the flask. On day 10, replace the medium with eight milliliters of enzyme-free EDTA-based cell dissociation buffer to collect the cells.
Incubate the cells briefly, then scrape the cells gently, and verify that they've released using a microscope. Once the cells are released, transfer the buffer with the cells to a 50 milliliter conical tube. Then rinse the flask three times with five milliliters of IMDM to collect more cells.
Pull all the collections, centrifuge them and resuspend the pellet in three milliliters of MCSF. Now, count the cells and adjust their concentration to one million per milliliter. Then plate 100 microliters of cell suspension per well in a 96-well flat bottom plate.
The entire plate should be fillable. Culture the cells for about an hour until they are adherent. Then stimulate them with different concentrations of LPS or intravenous immunoglobulin or both prepared in IMDM.
Perform all the manipulations in duplicate or triplicate and be certain to keep untreated controls. Then incubate the cells for 24 hours and analyze the supernatants. Prepare a one milliliter syringe with the required amount of intravenous immunoglobulin and LPS, and attach a 26-gauge needle.
For controls, use PBS plus LPS instead. Take hold of the mouse by its scruff while securing its hindquarters and tail using one hand. Target the lower-right quadrant of the abdomen, and insert the needle at a 30 to 40-degree angle with the bevel up, and insert it about 1.5 centimeters.
Then inject the bolus. After an hour, harvest peritoneal macrophages after euthanizing the mouse. In a sterile hood, pin the euthanized mouse to a foam board with limbs spread out and sterilize the abdomen with 70%ethanol.
Then, make a shallow two millimeter incision along the midline, and pull off the abdominal skin. Avoid puncturing the peritoneal wall. Next, fully load a five milliliter syringe with sterile PBS at pH 7.4, and inject the mouse using a 25 or 27-gauge needle, at the top of the peritoneal cavity, from either side, toward the midline, with the bevel of the needle up.
After pulling the needle out, massage the peritoneum for 10 seconds to dislodge the cells. Now, insert the needle again, back into the cavity, and collect about 3.75 milliliters of lavage fluid. Transfer the lavage fluid to a 15 milliliter conical tube on ice.
Repeat the injection, massage, and collection of lavage fluid three more times, and pull all the collections into the same tube. During the final collection it may be easier to withdraw the lavage fluid from the far side of the mouse. Next spin down the cells, and resuspend them in 500 microliters of plating medium.
Then count the viable macrophages, and resuspend them at one million per milliliter. Now, plate 100 microliters of suspension into the wells of a 96-well flat bottom plate. Next, incubate the cells for an hour.
The peritoneal macrophages will adhere to the plate. Then, remove the non-adherent cells, and rinse each well twice with 200 microliters of warm IMDM. After each wash, wait 10 seconds, and slowly tilt the plate to pull the rinse solution.
After the washes, add back 100 microliters of plating medium, and place the plate in the incubator. For the ELISA, continue to incubate the cells without stimulation for 24 hours. Then collect and clarify the conditioned medium for cytokine analysis.
BMDMs can be used to test antibody-based drug responses when challenged with an inflammatory stimulus in vitro. Intravenous immunoglobulin with LPS increases the production of the anti-inflammatory cytokine IL-10, seven-fold, compared to LPS stimulation alone. And it decreases the production of IL-12/23p40.
In addition, intravenous immunoglobulin stimulation alone or with LPS increased the activation of the MAP kinase ERK1/2, with early and prolonged phosphorylation. Activation of P-38 also occurred sooner. Furthermore, peritoneal macrophages significantly increased IL-10 production with stimulation in vitro, when compared to PBS-injected mice.
Cytokine production was next assessed in lavage fluid and in supernatants from ex vivo stimulated peritoneal macrophages. IL-10 was increased in lavage fluid, peritoneal cells, and peritoneal macrophages when the mice are challenged with intravenous immunoglobulin and LPS. By comparison, production of the pro inflammatory cytokine subunit IL-12/23p40 was significantly reduced in cultured peritoneal macrophages, but not in lavage fluid.
After watching this video, you should have a good understanding of how to test the effects of antibody-based drugs on macrophage activation states in vitro and in vivo using mouse bone marrow and peritoneal macrophages.
