The overall goal of this procedure is to observe the affects of notch signaling stimulation at varying points during the osteoclast differentiation process. This method can help answer key questions in the field of cell biology, such as:How does the role of notch signaling change during a cellular process? The main advantage of this technique is its marginality, which allows it to be adapted to other cells or notch ligands.

While this method can provide insight into osteoclast differentiation, it can also be applied to other processes such as cell survival, proliferation, or to the differentiation of other cell types. Begin the procedure by securing a mouse in the supine position and disinfecting the skin with 70 percent ethanol. Next, make an incision along the anterior surface of one hind limb to expose the tibia and femur.

Followed by an incision through the knee joint to free the distal end of the femur. Remove the femur by cutting through the end of the bone close to the pelvis and clear away any of the attached soft tissue. Cut through the ankle to remove the tibia and remove the soft tissue.

Then, use forceps to hold the leg bone over a 15 milliliter conical tube and insert a 25 and five-eighths gauged needle attached to a 10 milliliter syringe into the uncut end of the bone to flush two point five milliliters of room temperature alpha MEM through the cut end of the bone into the tube. When all of the bone marrow has been removed, the bone will turn a clear, beige color. After collecting the marrow from each bone, allow any large debris to settle to the bottom of the tube and transfer the debris-free, bone marrow cell containing supernatant into a new 15 milliliter tube.

Pellet the cells by centrifugation. Then lyse the red blood cells in zero point five milliliters of ACK Lysis buffer at 37 degrees Celsius for three minutes. At the end of the incubation, restore the isotonicity with 10 milliliters of PBS and collect the cells by centrifugation.

Resuspend the pellet in 10 milliliters of fresh Alpha MEM for plating. In the 100 milliliter tissue culture treated dish, promouse. And incubate the cells overnight at 37 degrees Celsius in a humidified tissue-culture incubator.

After enriching for the osteoclast precursors, wash the cells in five milliliters of PBS and treat them for three to five minutes with one milliliter of cell-dissociation reagent at room temperature until the osteoclast precursors can be dislodged with gentle tapping. Next, add four milliliters of Alpha MEM to the dish and transfer the cells to a 15 milliliter conical tube. After counting, dilute the precursors to a five times 10 to the fourth cells per milliliter concentration in Alpha MEM.

And treat them with MCSF and RANKL, then immediately seed one milliliter of cells into each well of a 24 well tissue-treated plate. Further incubation and 37 degrees Celsius in the humidified tissue culture incubator. After two days, replace the medium with one milliliter of fresh osteoclast medium to continue the differentiation.

For continuous notch signaling simulation, coat single well of a 24 well plate with 250 microliters of goat anti-human, IGG FC antibody in PBS. After one hour at room temperature, remove any unbound antibody with three washes and one milliliter of PBS. Next, add jagged1-FC fusion protein in PBS to the well for a two hour incubation at room temperature.

At the end of the incubation, wash the well three times with fresh PBS as just demonstrated. After the third wash, remove the PBS and immediately plate two point six times 10 to the fourth osteoclast precursors per centimeter squared onto the coated surface. Then transfer the plate to the tissue culture incubator for continuous notch signaling for up to three days.

For temporary notch signaling stimulation, first add one microgram of jagged1-FC with 40 microliters of protein G Agarose beads in one point five milliliters of PBS for two hour incubation with rotation at four degrees Celsius. At the end of the incubation, collect the beads by centrifugation followed by two washes and one point five milliliters of PBS under the same centrifuge conditions. After the second wash, re-suspend the jagged1 conjugated beads in 130 microliters per centimeter squared of culture medium and add the entire volume of beads directly onto two point six times 10 to the fourth osteoclast precursors in one well of a 24 well plate for their cold culture at 37 degrees Celsius and five percent carbon dioxide.

After the appropriate experimental stimulation period, remove the beads with several washes of PBS. Notch signaling activation can then be assayed by measuring the target gene transcription. When properly cultured, osteoclast precursors exhibit a primarily elongated, spindle-shaped morphology with a smooth cytoplasm.

Upon activation, the precursors spread and become flattened with foamy cytoplasm obtaining a resistance to RANK signaling and do not efficiently differentiate into mature osteoclasts. Under the correct culture and differentiation conditions, enriched osteoclast precursors will differentiate and infuse into large, TRAP-positive, multi-nuclear osteoclasts within three days of treatment. Seeding osteoclast precursors onto jagged1 FC-coated surfaces for their continuous stimulation induces the upper regulation of the expression of specific notch-target genes within 24 hours, while the expression of other notch target genes is not significantly altered by the stimulation.

Temporary notch signaling stimulation, via jagged1 FC coated beads with as little as 600 nanograms of jagged1 FC protein also induces a significant increase in the expression of the specific notch target genes. While attempting this procedure, it's important to remember that a common cause of osteoclast precursor differentiation failure is by accidental activation during handling. This can be assessed by measuring specific activation markers such as TNF to Inos.

After watching this video, you should have a good idea of how to differentiate osteoclast precursors and also to stimulate notch signaling, both transiently and continuously.