This work was supported by the National Natural Science Foundation of China (81473692) to Yong Ma. The authors thank all the staff of the Medical Research Center of the First College of Clinical Medicine in Nanjing University of Chinese Medicine.

All the methods involving the animals described here are approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing University of Chinese Medicine.
1. Setup
<ol>
	<li>Prepare several longitudinally cut 1 mL pipette tips (1 cm) and several 1.5 mL microcentrifuge tubes. Put the pipette tips and microcentrifuge tubes at 103 kPa and 121 &#176;C for 20 min and ensure that they are sterile.</li>
	<li>Prepare a box of ice and several sterile dishes to preserve the isolated tissues during the isolation procedure.</li>
</ol>
2. Preparation of Culture Medium
<ol>
	<li>Prepare the complete culture medium. Use minimum essential medium alpha (&#945;-MEM) that contains a final solution of 1% of penicillin/streptomycin and 10% fetal bovine serum (FBS).</li>
	<li>Filter the media from step 2.1 using a 0.22 &#956;m filter.</li>
	<li>Prepare the bone marrow induction medium. For 50 mL of the solution, add 125 &#181;L of M-CSF at a concentration of 10,000 ng/mL (Table of Materials) to 49.875 mL of complete culture medium (from step 2.2) to make a final solution of 25 ng/mL M-CSF.</li>
	<li>Prepare the osteoclast induction medium. For 50 mL of the solution, add 500 &#181;L of RANKL at a concentration of 10,000 ng/mL (Table of Materials) to 49.5 mL of bone marrow induction medium (from step 2.3) to make a final solution of 25 ng/mL M-CSF and 100 ng/mL RANKL.</li>
</ol>
3. Isolation of Bone mMarrow-derived Cells
<ol>
	<li>Euthanasia
	<ol>
		<li>Euthanize four Sprague Dawley rats by CO2 inhalation followed by cervical dislocation, and immerse them in 75% ethanol for 1 min. 
		NOTE: Ensure that animals are of the same sex and of a similar age. Animals that are 2 to 3 weeks old are recommended. Two rats are prepared for the centrifugal method, while the other two are for the traditional method.</li>
	</ol>
	</li>
	<li>Traditional method
	<ol>
		<li>Place the animals on a disinfectant board in a supine position. Make a small incision (approximately 1 cm) at the proximal femur to peel the skin, using sterile scissors.</li>
		<li>Dissect out the femurs and tibias. Cut the bilateral connection parts around the hip, knee, and ankle joints to isolate the tibias and femurs carefully and gently. Cut off part of the tissues around the bone; be thorough. Do not fracture the tibias and femurs during the whole process.</li>
		<li>Place the cleaned bones into a dish with 5 mL of the complete culture medium. Cut off the long bone with sterile scissors and use a 1 mL syringe needle to flush the marrow cavity carefully with 10 mL of complete media until the marrow cavity turns white.</li>
		<li>Transfer the cell suspension from step 3.2.3 to a 50 mL tube, and then, filter it with a 70 &#956;m strainer to remove the remaining tissue.</li>
		<li>Add 5 mL of red blood cell lysis buffer (Table of Materials) to the cell suspension. Incubate the cells for 8 min on ice. Then, centrifuge the cells at 250 x g for 5 min to yield the cell pellet.</li>
		<li>Aspirate off the medium and resuspend the cells in 10 mL of complete media. Count the cells using a hemocytometer and calculate the time spent on the steps from this section (section 3.2).</li>
	</ol>
	</li>
	<li>Improved method
	<ol>
		<li>Place the animals on a disinfectant board in a supine position. Make a small incision (approximately 1 cm) at the proximal femur with sterile scissors to peel the skin.</li>
		<li>Dissect out the femurs and tibias. Properly cut off the part of the tissues around the bone (no need to remove completely). Do not fracture the tibias and femurs during the whole process.</li>
		<li>Rinse the tibias and femurs with 12 mL of phosphate-buffered saline (PBS) and cut them in half. Place the snipped tibias and femurs into a 1 mL pipette tip (from step 1.1), which is then put into a microcentrifuge tube and is centrifuged 3x at 1,000 x g for 45 s at 4 &#176;C.</li>
		<li>After centrifugation, remove the pipette tip containing the bone, and leave the bone marrow in the tube. Add 200 &#956;L of complete media into the microcentrifuge tube and repeat pipetting to disintegrate the marrow thoroughly.</li>
		<li>Transfer the cell suspension from step 3.3.4 to a 50 mL tube, and then, filter it with a 70 &#956;m strainer to remove the remaining tissue.</li>
		<li>Count the cells using a hemocytometer, and calculate the time spent on the steps from this section (section 3.3).</li>
	</ol>
	</li>
</ol>
4. Purification by Density Gradient Centrifugation
<ol>
	<li>Adjust the cell suspension from step 3.2.6 or step 3.3.5 to 2 mL with &#945;-MEM.</li>
	<li>Prepare one sterile silicified centrifugal tube and add 8 mL of the cell separation solution (Table of Materials) to the tube.</li>
	<li>Add the cell suspension from step 4.1 to the cell separation solution. Adhere the pipette tip to the inner surface of the tube and keep a 45&#176; to the inner surface. After adding the suspension, a clear limit will appear between the layer of the cells and the separation solution. 
	NOTE: The operation requires great care and needs to be performed slowly.</li>
	<li>Centrifuge the layered solution in a horizontal centrifuge at 500 x g for 30 min. After centrifugation, aspirate off the cloudy second layer, which contains the target cells, from top to bottom. 
	NOTE: Cancel the acceleration and deceleration of the centrifuge before centrifugation. There are six layers after centrifugation; the first layer is the diluent layer, the second layer is the monocytes layer, the third layer is the layer of mononuclear cells, the fourth layer is the transparent separation liquid one layer, the fifth is the granular cell layer, and the sixth layer is the red cell layer.</li>
	<li>Transfer the target cells to a new tube. Add 5 mL of PBS to wash the cells 3x. After each wash, centrifuge the target cells at 250 x g for 5 min to yield the cell pellet.</li>
	<li>Resuspend the cell pellet with bone marrow induction medium and count the cells using a hemocytometer. Add 5&#8211;8 mL of bone marrow induction medium from step 2.3 to obtain a final cell solution of 300,000 cells/mL. Add 1 mL to each well of a 24-well plate.</li>
</ol>
5. Culture and Differentiation
<ol>
	<li>After incubating the cells at 37 &#176;C for 24 h, gently aspirate off the medium and add 1 mL of the osteoclast induction medium to each well. Agitate the plate gently and put it in an incubator at 37 &#176;C.</li>
	<li>Change the osteoclast induction medium every 48 h. While doing so, change 0.8 mL of the medium in the well and agitate the plate gently. 
	NOTE: Large, motile, and multinucleated osteoclasts should be observed in the well under an inverted microscope, typically around days 4&#8211;6.</li>
</ol>
6. Tartrate-resistant Acid Phosphatase Staining
NOTE: Multinucleate osteoclasts will be present after 4&#8211;6 days if the induction (section 5) is successful.
<ol>
	<li>Prepare the fixative solution by combining 4 mL of 37% formaldehyde, 32.5 mL of acetone, and 12.5 mL of a citrate solution. Store the fixative solution at 4 &#176;C.</li>
	<li>Prepare the tartrate-resistant acid phosphatase (TRAP) stain solution (Table of Materials) by adding 50 &#181;L of sodium nitrite, 50 &#181;L of Fast Garnet GBC base solution, 50 &#181;L of naphthol AS-BI phosphate solution, 200 &#181;L of an acetate solution, and 100 &#181;L of a tartrate solution into 4.55 mL of deionized water that is prewarmed to 37 &#176;C. Then, mix gently by inversion for 1 min, and let it stand for 2 min.</li>
	<li>After the successful induction of osteoclasts, aspirate off the medium, and gently wash the wells 3x with PBS.</li>
	<li>Bring the fixative solution to room temperature (RT). Add 2 mL of fixative solution to the wells for 30 s, and do not allow the cells to dry.</li>
	<li>Aspirate the fixative solution, gently wash it 3x with deionized water that is prewarmed to 37 &#176;C, and then, aspirate the water.</li>
	<li>Add 2 mL of TRAP stain solution for 1 h at 37 &#176;C and keep the sample in the dark.</li>
	<li>After 1 h, aspirate the stain, and gently wash the sample with deionized water that is prewarmed to 37 &#176;C, and then, aspirate the water.</li>
	<li>Counterstain the cells for 1 min in a hematoxylin solution. After staining, aspirate the hematoxylin solution and gently wash the cells 3x with deionized water.</li>
	<li>Image osteoclasts using brightfield microscopy, and TRAP+ cells with three or more nuclei will appear purple.</li>
</ol>
7. Bone Resorption Assay Using Toluidine Blue Staining
<ol>
	<li>Pretreatment of the bone slices
	<ol>
		<li>Cut the fresh bovine femoral bone cortex into 2 cm thick slices along the longitudinal axis by microelectric saw. Then, cut and grind the slices with hard tissue grinders into 80 &#181;m.</li>
		<li>Wash the slices in a beaker with deionized water and 100 Hz ultrasound for 1 h, and repeat 3x.</li>
		<li>Immerse the slices into 75% alcohol for 2 h. Then, aspirate off the alcohol and expose each side of the slices to ultraviolet light for 1 h on a clean platform.</li>
		<li>Before planting the cells, immerse the slices in the culture medium for at least 2 h.</li>
	</ol>
	</li>
	<li>Toluidine blue staining
	<ol>
		<li>Place the pretreated bone slices into the 24-well plate. Plant the cells as mentioned in step 4.6 and induce the cells as mentioned in section 5.</li>
		<li>After the appearance of osteoclasts (after 4&#8211;6 days), wash the slices with 1 mL of 0.25 M ammonium hydroxide, and sonicate them 3x for 5 min each to remove the living cells to allow the analysis of the resorption pits on the bone slices. Then, remove the ammonium hydroxide and stain the slices with 1 mL of 1% (wt/vol) toluidine blue solution per slice for 2 min.</li>
		<li>Wash the slices with PBS. Randomly select five views and perform a semiquantitative analysis of the resorption area using an image analysis software.</li>
	</ol>
	</li>
</ol>
8. Scanning Electron Microscopy
<ol>
	<li>Prefix the bone slices from step 7.2.1 with 1 mL of 2.5% glutaraldehyde per slice for 2 h at RT, and then, sonicate the slices 3x for 3 min each with 1 M ammonium hydroxide to remove the cells and remove the ammonium hydroxide.</li>
	<li>Wash the bone slices 3x for 12 min each with PBS.</li>
	<li>Fix the bone slices with 1% osmic acid for 2 h at RT.</li>
	<li>Perform ethanol gradient dehydration, with 50%, 70%, 80%, 90%, and 95% ethanol, for 15 min for every gradient, and then, replace the ethanol with isoamyl acetate for 15 min.</li>
	<li>Coat the slices with gold palladium, and then, analyze by scanning electron microscopy.</li>
</ol>
9. Immunofluorescence Staining of Calcitonin Receptor
NOTE: Multinucleate osteoclasts will be present after 4&#8211;6 days if the induction is successful (section 5).
<ol>
	<li>Aspirate off the medium and gently wash the well 3x with PBS.</li>
	<li>Fix the cells for 10 min with 4% paraformaldehyde, which is precooled to 4 &#176;C.</li>
	<li>Add 1 mL of PBS with 0.3% nonionic surfactant per well for 30 min on ice. Then, aspirate off the PBS with 0.3% nonionic surfactant and add 1 mL of PBS with 5% FBS per well for 30 min on ice.</li>
	<li>Aspirate off the PBS and incubate the membranes with anti-calcitonin receptor (anti-CTR) at a 1:100 dilution in PBS at 4 &#176;C overnight.</li>
	<li>Aspirate off the solution, and incubate the membranes with Alexa-488-conjugated anti-rabbit IgG antibodies at a 1:1,000 dilution in PBS for 1 h at RT.</li>
	<li>Aspirate off the solution and gently wash the cells 3x with PBS. Counterstain the nuclei with Hoechst 33342 stain for 3 min at RT.</li>
	<li>Aspirate off the solution, and gently wash the cells 3x with PBS. Observe the intensity of CTR by fluorescence microscopy.</li>
</ol>

<ol>
	<li>Boyle, W. J., Simonet, W. S., Lacey, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclast+differentiation+and+activation.">Osteoclast differentiation and activation.</a> Nature. 423 (6937), 337-342 (2003).</li><li>Kular, J., Tickner, J., Chim, S. M., Xu, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+the+regulation+of+bone+remodelling+at+the+cellular+level.">An overview of the regulation of bone remodelling at the cellular level.</a> Clinical Biochemistry. 45 (12), 863-873 (2012).</li><li>Park, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+of+the+reduced+enamel+epithelium+and+its+implications+for+bone+resorption+during+tooth+eruption.">Apoptosis of the reduced enamel epithelium and its implications for bone resorption during tooth eruption.</a> Journal Of Molecular Histology. 44 (1), 65-73 (2013).</li><li>Boyde, A., Ali, N. N., Jones, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resorption+of+dentine+by+isolated+osteoclasts+in+vitro.">Resorption of dentine by isolated osteoclasts in vitro.</a> British Dental Journal. 156 (6), 216-220 (1984).</li><li>Chambers, T. J., Revell, P. A., Fuller, K., Athanasou, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resorption+of+bone+by+isolated+rabbit+osteoclasts.">Resorption of bone by isolated rabbit osteoclasts.</a> Journal of Cell Science. 66, 383-399 (1984).</li><li>Takahashi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclast-like+cell+formation+and+its+regulation+by+osteotropic+hormones+in+mouse+bone+marrow+cultures.">Osteoclast-like cell formation and its regulation by osteotropic hormones in mouse bone marrow cultures.</a> Endocrinology. 122 (4), 1373-1382 (1988).</li><li>Takahashi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoblastic+cells+are+involved+in+osteoclast+formation.">Osteoblastic cells are involved in osteoclast formation.</a> Endocrinology. 123 (5), 2600-2602 (1988).</li><li>Suda, T., Takahashi, N., Martin, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+osteoclast+differentiation.">Modulation of osteoclast differentiation.</a> Endocrine Reviews. 13 (1), 66-80 (1992).</li><li>Yoshida, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+murine+mutation+osteopetrosis+is+in+the+coding+region+of+the+macrophage+colony+stimulating+factor+gene.">The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene.</a> Nature. 345 (6274), 442-444 (1990).</li><li>Yasuda, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclast+differentiation+factor+is+a+ligand+for+osteoprotegerin/osteoclastogenesis-inhibitory+factor+and+is+identical+to+TRANCE/RANKL.">Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL.</a> Proceedings of The National Academy of Sciences of The United States of America. 95 (7), 3597-3602 (1998).</li><li>Lacey, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoprotegerin+ligand+is+a+cytokine+that+regulates+osteoclast+differentiation+and+activation.">Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation.</a> Cell. 93 (2), 165-176 (1998).</li><li>Marino, S., Logan, J. G., Mellis, D., Capulli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+culture+of+osteoclasts.">Generation and culture of osteoclasts.</a> BoneKEy Reports. 3, 570 (2014).</li><li>Pei, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluoride+decreased+osteoclastic+bone+resorption+through+the+inhibition+of+NFATc1+gene+expression.">Fluoride decreased osteoclastic bone resorption through the inhibition of NFATc1 gene expression.</a> Environmental Toxicology. 29 (5), 588-595 (2014).</li><li>Arnett, T. R., Dempster, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+pH+on+bone+resorption+by+rat+osteoclasts+in+vitro.">Effect of pH on bone resorption by rat osteoclasts in vitro.</a> Endocrinology. 119 (1), 119-124 (1986).</li><li>Arnett, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+is+a+major+stimulator+of+osteoclast+formation+and+bone+resorption.">Hypoxia is a major stimulator of osteoclast formation and bone resorption.</a> Journal of Cellular Physiology. 196 (1), 2-8 (2003).</li><li>Orriss, I. R., Arnett, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+osteoclast+cultures.">Rodent osteoclast cultures.</a> Methods in Molecular Biology. 816, 103-117 (2012).</li><li>Quinn, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcitonin+receptor+antibodies+in+the+identification+of+osteoclasts.">Calcitonin receptor antibodies in the identification of osteoclasts.</a> Bone. 25 (1), 1-8 (1999).</li></ol>

The authors have nothing to disclose.

The ability to obtain and study osteoclasts in vitro is a critical and fundamental skill for any researcher wishing to study bone metabolism, which may help understand the mechanisms of bone-absorbing diseases and develop novel therapeutic agents. The present study described a protocol with some modifications based on previous methods.
By using pipette tips and microcentrifuge tubes to obtain bone marrow, it largely reduced the operation time of obtaining bone marrow and the workload of labora...

Bone homeostasis is a complex physiological process that is regulated by bone-resorbing osteoclasts and bone-forming osteoblasts1. A balance between osteoblastic and osteoclastic activity mediated through osteoblasts and osteoclasts, respectively, is highly essential for maintaining bone health and homeostasis, because perturbations in bone homeostasis might lead to bone diseases, such as abnormal bone growth or loss of bone density. As unique bone-resorption cells, osteoclasts are important in diseases related to abnormal bone destruction, including osteoporosis, periodontitis, and periprosthetic osteolysis2,3.
The development of an osteoclast culture is mainly divided into two stages. The methods established by Boyde&#160;et al.4 and Chambers&#160;et al.5 composed the first stage in the 1980s. They obtained relatively abundant osteoclasts from the bones of newborn animals, which is the rapid remodeling period. Osteoclasts are released by fragmenting and stirring the bones in a special medium. However, the cells obtained by this method are low in quantity and purity. The second stage was the development of long-range cultures of osteoclast formation, using hematopoietic lineage cells derived from bone marrow6. Cytokines, such as 1&#945;,25-dihydroxyvitamin D3, prostaglandin E2 (PGE-2), and parathyroid hormone (PTH), which are added into the culture medium, act through the system of osteoblasts/stromal cells to stimulate osteoclast formation7,8. However, the purity and quantity of osteoclasts obtained by this method cannot meet the needs of modern molecular biology research. Then, the discovery of macrophage colony-stimulating factor (M-CSF) and receptor activator for nuclear factor-&#954;B ligand (RANKL) make osteoclastogenesis easier9,10,11, and the method of using M-CSF and RANKL to directly stimulate osteoclast formation is widely used around the world. However, there are still some details in the methodology that need to be improved.
Currently, the most commonly used osteoclast culture method, as described by Marino et al.12 and Pei et al.13, often requires the removal of the surrounding tissue around the bone and uses a sterilized needle to flush the marrow cavity with completed media. There are some drawbacks to this process, including the fact that (1) the removal of the surrounding tissue around the bone requires much time and great surgical technique, (2) the bones are fragile and can lead to bone marrow outflow, (3) the bone marrow cavity might be too tiny to flush, and (4) there is a risk of needle stick injury. To avoid these problems, we centrifuge the tubes containing the bones for bone marrow instead of needle-flushing the bone marrow. Here, we introduce a stable and safe method which isolates bone marrow in less time and with less effort compared to the traditional procedure. Together with the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts&#160;in vitro.

<table>
	<tbody>
		<tr>
			<td>Acid Phosphatase, Lekocyte (TRAP) kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A</td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic Hard Tissue Slicer</td>
			<td>Lecia</td>
			<td>RM2265</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine femoral bone</td>
			<td></td>
			<td></td>
			<td>Purchased by ourselves</td>
		</tr>
		<tr>
			<td>Cell Incubator</td>
			<td>Heraus</td>
			<td>BB16/BB5060</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sarana</td>
			<td>s-fbs-au-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG H&#38;L (Alexa Fluor 488)</td>
			<td>Abcam</td>
			<td>ab150077</td>
			<td></td>
		</tr>
		<tr>
			<td>Hard Tissue Grinders</td>
			<td>Lecia</td>
			<td>SP-2600</td>
			<td></td>
		</tr>
		<tr>
			<td>Histopaque Kit</td>
			<td>TianJing Haoyang</td>
			<td>TBD2013DR</td>
			<td>Silicified centrifugal tube amd cell separation solution</td>
		</tr>
		<tr>
			<td>Hochest33342</td>
			<td>Sigma-Aldrich</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Phase Contrast Microscope</td>
			<td>Olympus</td>
			<td>CKX31</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescence Microscope</td>
			<td>Lecia</td>
			<td>DMI-3000</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM, no Glutamine</td>
			<td>Gibco</td>
			<td>11090-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin, Liquid</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Anti-Calcitonin receptor</td>
			<td>Bioss</td>
			<td>bs-0124R</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Rat M-CSF</td>
			<td>PeproTech</td>
			<td>400-28</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Rat sRANK Ligand</td>
			<td>PeproTech</td>
			<td>400-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysis Buffer</td>
			<td>Absin</td>
			<td>abs47014932</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscopy</td>
			<td>FEI</td>
			<td>Quanta 200</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine Blue</td>
			<td>Sigma-Aldrich</td>
			<td>89649</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, purification, and differentiation of osteoclast precursors from rat bone marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

The purpose of the protocol was to isolate and purify large numbers of osteoclast precursors conveniently and induce osteoclasts successfully. By supplementing with M-CSF and RANKL, giant osteoclasts were seen on days 5&#8211;6. The formation of osteoclasts was successfully identified by TRAP staining (Figure 1A). Large and purple cells were regarded as TRAP-positive cells with multiple nuclei (typically &#8805; three nuclei). Through this method, it was typi...

Watch this Scientific Journal Video about Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow at JoVE.com

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

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JoVE Journal

Biology

10.0K Views.  Nanjing University of Chinese Medicine. Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Video: Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...