The overall goal is this procedure is to generate and distinguish alveolar-like macrophages from dendritic cells in in vitro murine bone marrow cell cultures supplemented with GM-CSF. This in vitro method can help answer key questions regarding the function of dendritic cells or macrophages. The main advantage of the technique is that it can generate sufficient cell numbers and it can distinguish between dendritic cells and alveolar-like macrophages.
The implications of this technique may extend towards the therapy of pulmonary proteinosis as it facilitates the acquisition of alveolar-like macrophages, which can be used to treat this disease. Generally, individuals new to this method will need practice with isolating the cleaned bones and flushing the bone marrow. Visual demonstration of this method is useful as not all techniques used as easily understood by written instruction alone.
15 minutes before beginning the dissection, turn on a biological safety cabinet to purge the cabinet air, and to stabilize the air flow, and clean the cabinet surface with 70%ethanol. When the cabinet is ready, place a mouse on top of one to two paper towels in the prone position and spray the animal with 70%ethanol. Then, with the non-dominant hand, lift the skin around the thoracic area and use scissors to make an incision.
Grasping both ends of the incision, carefully pull until the two ends of the incision meet at the stomach. Then place the mouse in the supine position and use one hand to pull the lower half of the skin down until the legs are exposed. Holding the foot, cut the Achilles tendons above the ankle joint and the ligaments connecting the muscles to the foot at the lower end of the tibia to loosen the foot from the leg bones.
Keeping the leg raised, remove the muscles and ligaments around the knee joint at the lower end of the femur to severe the thigh muscles from the lower leg. Using forceps, gently pull the loosened muscles away from the fibular and femoral surfaces. Then pressing the scissors in the open position at the hip joint, rotate the leg and gently pull on the femur to separate the leg bone from the hip joint while cutting the leg free from the body.
Now holding the leg at the hip end with a sterile forceps, use another forceps to remove the foot from the ankle end. Then holding the distal end of the femur with one forceps and the proximal end of the tibia and fibula with another, carefully bend the tibia and fibula in the opposite direction of the knee joint to separate the lower leg from the femur and patella. Use the forceps to remove the remaining ligaments, muscles, and fibula from the lower leg bones and place the cleaned tibia on an inverted Petri dish lid, containing one to two milliliters of HBSS/FCS.
Holding the lower end of the femur with one set of forceps and the patella with another, bend the knee and surrounding tissues in the opposite direction to remove the joint. Then remove any of the remaining connective tissues from the femur and place the bone on the inverted Petri dish lid. When all of the bones have been harvested, use forceps to clean any residual tissue still attached to the bones and immerse the bones in 10 milliliters of HBSS/FCS within the base of the Petri dish.
Keeping the dish partially shielded with a sterile lid, cut each of the cleaned bones in half with scissors. Then use a one-milliliter syringe, equipped with a 26.5-gauge needle to flush one milliliter of HBSS/FCS from the dish into the end of each bone piece until all of the red marrow has been liberated. Pass any visible clumps of bone marrow through the syringe several times to form a single cell suspension, followed by thorough mixing with a 10 milliliter pipette.
Transfer the cells to a collection tube and rinse the Petri dish one time with five milliliters of HBSS/FCS to collect any residual cells. Then pool the wash in the collection tube and place the cells on ice. To set up a bone marrow cell culture, centrifuge the collected cells and aspirate the supernatant with a sterile glass Pasteur pipette attached to vacuum suction.
Loosen the red blood cell-rich pellet with tapping. Then add 10 milliliters of RBC lysis buffer and resuspend the cells with vortexing. After a five-minute, room temperature incubation, arrest the lysis with 10 milliliters of HBSS/FCS and spin down the cells, resuspending the pellet in bone marrow cell medium.
Count the number of viable cells by Trypan Blue exclusion and transfer the needed cells into a new tube with bone marrow cell medium. Then collect the cells by centrifugation and resuspend the pellet at a four million cell per milliliter dilution in bone marrow cell medium with 20 nanograms per milliliter of GM-CSF. Next, add 9.5 milliliters of bone marrow cell medium supplemented with 20 nanograms per milliliter of GM-CSF to one sterile bacterial Petri dish per culture and add 500 microliters of cells to the center of each dish.
Incubate the cells at 37 degrees Celsius and five percent CO2. On day three, add 10 milliliters of fresh bone marrow medium supplemented with 20 nanograms per milliliter of GM-CSF to each culture, taking care to minimize any disturbances to the cells. On day six, carefully transfer 10 milliliters of cell culture supernatant to a sterile 50 milliliter conical tube.
Centrifuge the cells and resuspend the pellet in 10 milliliters of fresh bone marrow cell medium, containing 20 nanograms per milliliter of GM-CSF. Then gently vortex the cell suspension and return the cells to the culture dish. On day one, the bone marrow cells are small and sparse but by day three, the cells have increased in number and size, and some have begun to adhere.
By day six, there are more cells and both adherent and non-adherent fractions are observed. The culture can be harvested from day seven to 10 and gated by size and live cells with a higher percentage of CD11c-positive cells obtained from day 10 cultures. At day seven, the collected non-adherent fraction is greatly enriched for cells with a dendritic morphology.
The CD11c-positive, GR1-negative populations, harvested on day seven and 10, can be divided into three main populations. Macrophages, immature dendritic cells, and mature dendritic cells by MHCII expression and FL-HA binding. Interestingly, MerTK, although considered to be a macrophage marker, exhibits a robust expression on both macrophage and immature dendritic cell populations, harvested on days seven and 10, with a low level of expression observed on mature dendritic cells.
While attempting this procedure, it is important to remember to use sterile techniques throughout the bone marrow harvest and cell culture. Following this procedure, the cells also can be sorted to examine cell populations separately, or they can be stimulated with inflammatory agents and subsequently analyzed for their cytokine production by intracellular staining and flow cytometry. After watching this video, you should have a good understanding of how to isolate bone marrow cells and culture them in GM-CSF to generate both dendritic cells and alveolar-like macrophages.
