Stimulation of Notch Signaling in Mouse Osteoclast Precursors

All research involving vertebrate animals was performed in accordance with protocols approved by the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC).

1. Culture Media Preparation

1. Prepare α-MEM by dissolving minimum essential medium (MEM) powder and 1.9 g sodium bicarbonate in 900 ml H₂O and supplement with 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 100 ml heat-inactivated fetal bovine serum. Sterilize using a 0.22 µm polyethersulfone (PES) filter.
2. Prepare macrophage medium by supplementing α-MEM with 35 ng/ml recombinant mouse Macrophage-Colony Stimulating Factor (M-CSF)
3. Prepare osteoclast medium by supplementing macrophage medium with 100 ng/ml RANKL

2. Bone Marrow Cell Isolation

1. Fill one 10 ml syringe with a 25⅝ gauge needle per mouse with 10 ml α-MEM.
2. Euthanize mice via 10 min exposure to CO₂ at a flow rate of 1.3 L/min. Ensure animal death by following CO₂ exposure with cervical dislocation before proceeding with dissection.
3. Using clean technique, dissect out and separate the tibiae and femora. Disinfect mouse skin with 70 % ethanol before making an incision along the anterior surface of each hind limb to expose the tibia and femur.
   1. Cut through the knee joint to free the distal end of the femur, and cut through the femur as close to the pelvis as possible. Remove the femur and clean as much soft tissue as possible. To remove the tibia, cut through the ankle and remove as much soft tissue as possible.
4. Hold the bone with forceps and flush marrow into a 15 ml conical tube with 2.5 ml room temperature α-MEM combining marrow flushes from a single mouse into a single tube.
   NOTE: A successful marrow flush will change the coloration of the bone from red to beige.
5. Allow debris to settle to the bottom of the tube and transfer supernatant to a clean 15 ml conical tube taking care to avoid transferring any debris.
   NOTE: Some cells may also settle with the marrow debris. If a greater number of cells is required, marrow flushes can be filtered through a 70 µm cell strainer into a clean 15 ml tube.
6. Centrifuge tube(s) at 300 x g for 5min at room temperature to pellet cells.
7. Vacuum aspirate the supernatant and resuspend cell pellet in 0.5 ml Ammonium-Chloride-Potassium (ACK) lysing buffer and incubate at 37 °C for 3 min to lyse red blood cells.
8. Add 10 ml PBS directly to ACK-cell solution, and centrifuge at 300 x g for 5 min.
   NOTE: The previously red cell pellet should now be beige.
9. Vacuum aspirate supernatant and resuspend pellet in 10 ml α-MEM, and plate in a 100 mm tissue culture-treated dish. Incubate cells overnight at 37 °C in a humidified tissue culture incubator.
   NOTE: Counting cells is not necessary for this step. During this time, adherent cells from the bone marrow will attach to the culture surface; non-adherent bone marrow cell populations will remain in suspension. MCSF is not present in the culture medium during this initial incubation.

3. Enrichment of Osteoclast Precursors

1. After overnight incubation of marrow flushes, transfer culture supernatant containing non-adherent cells to a 50 ml conical tube.
   NOTE: Culture dishes with adherent cells, which include bone marrow mesenchymal progenitor cells, can be discarded or fed with fresh α-MEM, if desired for other experiments.
   NOTE: Osteoclasts can be differentiated directly from these non-adherent bone marrow cells by plating them into tissue culture-treated dishes at a density of 4 x 10⁵ cells/cm² in osteoclast medium and refreshing medium every other day for 5 days.
2. Add α-MEM to non-adherent cell solution to a final volume of 18 ml and add M-CSF to a final concentration of 35 ng/ml.
3. Add 3 ml of cell suspension each to 6 60 mm suspension culture dishes.
4. Incubate plates overnight at 37 °C in a humidified tissue culture incubator. During this time, M-CSF will stimulate differentiation of macrophages, which can adhere even to non-tissue culture-treated surfaces.
5. After incubating overnight, re-feed osteoclast precursors with 3 ml fresh macrophage medium. This will remove non-adherent cells that haven't differentiated into macrophages.
6. Continue to culture osteoclast precursors at 37 °C and 5 % CO₂ for 2 days or until cultures reach roughly 70% confluence.

4. Osteoclast Differentiation

1. Wash osteoclast precursors with 5 ml PBS and treat cells with 1 ml marine-origin proteolytic/collagenolytic enzyme at room temperature until the cells can be dislodged by gentle tapping of the plate.
   NOTE: Osteoclast precursors can only be lifted if they are cultured in suspension dishes; precursors attach too firmly to tissue culture-treated surfaces to be lifted. Trypsin can activate osteoclast precursors and should be avoided.
2. Add 4 ml α-MEM to each dish and transfer cells to a 50 ml conical tube. Count cells using a hemocytometer and dilute cells to a density of 5 x 10⁴ cells/ml and add M-CSF to a final concentration of 35 ng/ml and RANKL to a final concentration of 100 ng/ml.
3. Seed cells at a density of 2.6 x 10⁴ cells/cm² into tissue culture-treated plates and incubate at 37 °C in a humidified tissue culture incubator for 2 days. Differentiation is typically carried out in 24-well plates, where 5 x 10⁴ cells are seeded into each well.
4. 2 days after the start of the differentiation, re-feed cells by replacing medium with 1 ml fresh osteoclast medium.
   NOTE: Osteoclast differentiation will usually be complete within 3 days. Resulting osteoclasts can be TRAP-stained for easy quantification and morphological analysis.

5. Continuous Stimulation of Notch Signaling with Jagged1-coated Surface

1. Suspend goat anti-human IgG Fc antibody in PBS at a concentration of 10 μg/ml (1:1,000 dilution of 10 mg/ml stock). Add antibody solution to culture surface at a density of 1.3 μg/cm² and incubate at room temperature for 1 hr. In the case of 24-well plates, add 250 μl (2.5 μg) per well.
2. Vacuum aspirate wells and refill with 1 ml PBS. Repeat this step 2 times.
3. Suspend recombinant Jagged1-Fc fusion protein in PBS at a concentration of 10 μg/ml. Add Jagged1-FC solution to the antibody-coated culture surface at a density of 1.3 μg/cm² and incubate at room temperature for 2 hr. Culture surfaces coated with antibody only can be used as controls.
4. Vacuum aspirate wells and refill with 1 ml PBS. Repeat this step 2 times. Keep PBS in wells until just prior to cell seeding to prevent them from drying.
5. Seed 2.6 x 10⁴ osteoclast precursors/cm² onto coated surfaces. Notch signaling will be continuously stimulated for at least 3 days.

6. Temporary Stimulation of Notch Signaling with Jagged1-coated Beads

1. Combine the following in an appropriately sized tube: 0.5 μg/cm² Jagged1-Fc, 21 μl/cm² protein G agarose beads, and PBS to a final volume of 1.5 ml. To prepare an adequate number of Jagged1 beads for 1 well of a 24 well plate, combine 1 μg Jagged1-Fc, 40 μl protein G agarose beads, and PBS to 1.5 ml.
2. Incubate tube with rotation at 4 °C for 2 hr.
3. Centrifuge tube at 300 x g for 1 min to pellet beads. Resuspend beads in 1.5 ml PBS. Repeat this wash 2 more times.
4. Resuspend Jagged1 beads in 130 μl/cm² culture medium and apply to cultured cells.
5. Incubate cells with beads at 37 °C and 5 % CO₂ for desired period. Remove beads with several washes of PBS or culture medium.
6. Verify Notch signaling activation via measurement of Notch target gene transcripts¹⁰.