This work was partially funded by the China Scholarship Council [CSC No. 201808440394]. W.C. was financed by CSC.

The protocol was reviewed and approved by the local ethics committee (approval number 287/2020B02).
1. Preparation of calcium phosphate coated cell culture plates
<ol>
	<li>Preparation of calcium stock solution (25 mM CaCl2&#183;2H2O, 1.37 M NaCl, 15 mM MgCl2&#183;6H2O in Tris buffer)
	<ol>
		<li>Prepare 50 mM Tris buffer and adjust pH to 7.4 using 1 M HCl.</li>
		<li>Set up a glass beaker on a magnetic stirrer and add 100 mL of 50...

<ol>
	<li>Jacome-Galarza, C. E., 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=Developmental+origin,+functional+maintenance+and+genetic+rescue+of+osteoclasts.">Developmental origin, functional maintenance and genetic rescue of osteoclasts.</a> Nature. 568 (7753), 541-545 (2019).</li><li>Moller, A. M. 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=Aging+and+menopause+reprogram+osteoclast+precursors+for+aggressive+bone+resorption.">Aging and menopause reprogram osteoclast precursors for aggressive bone resorption.</a> Bone Research. 8 (1), 1-11 (2020).</li><li>Yokota, K., 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=Characterization+and+function+of+tumor+necrosis+factor+and+interleukin-6-induced+osteoclasts+in+rheumatoid+arthritis.">Characterization and function of tumor necrosis factor and interleukin-6-induced osteoclasts in rheumatoid arthritis.</a> Arthritis Rheumatology. 73 (7), 1145-1154 (2021).</li><li>Teng, Y. T., 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=Functional+human+T-cell+immunity+and+osteoprotegerin+ligand+control+alveolar+bone+destruction+in+periodontal+infection.">Functional human T-cell immunity and osteoprotegerin ligand control alveolar bone destruction in periodontal infection.</a> Journal of Clinical Investigation. 106 (6), 59-67 (2000).</li><li>Terpos, E., 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=Soluble+receptor+activator+of+nuclear+factor+kappaB+ligand-osteoprotegerin+ratio+predicts+survival+in+multiple+myeloma:+proposal+for+a+novel+prognostic+index.">Soluble receptor activator of nuclear factor kappaB ligand-osteoprotegerin ratio predicts survival in multiple myeloma: proposal for a novel prognostic index.</a> Blood. 102 (3), 1064-1069 (2003).</li><li>Morony, S., 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+inhibits+osteolysis+and+decreases+skeletal+tumor+burden+in+syngeneic+and+nude+mouse+models+of+experimental+bone+metastasis.">Osteoprotegerin inhibits osteolysis and decreases skeletal tumor burden in syngeneic and nude mouse models of experimental bone metastasis.</a> Cancer Research. 61 (11), 4432-4436 (2001).</li><li>Sobacchi, C., Schulz, A., Coxon, F. P., Villa, A., Helfrich, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteopetrosis:+genetics,+treatment+and+new+insights+into+osteoclast+function.">Osteopetrosis: genetics, treatment and new insights into osteoclast function.</a> Nature Reviews Endocrinology. 9 (9), 522-536 (2013).</li><li>Amarasekara, D. S., 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=Regulation+of+osteoclast+differentiation+by+cytokine+networks.">Regulation of osteoclast differentiation by cytokine networks.</a> Immune Network. 18 (1), 8 (2018).</li><li>Kim, J. M., Lin, C., Stavre, Z., Greenblatt, M. B., Shim, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoblast-osteoclast+communication+and+bone+homeostasis.">Osteoblast-osteoclast communication and bone homeostasis.</a> Cells. 9 (9), (2020).</li><li>Teitelbaum, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+resorption+by+osteoclasts.">Bone resorption by osteoclasts.</a> Science. 289 (5484), 1504-1508 (2000).</li><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>Baron, R., Neff, L., Louvard, D., Courtoy, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-mediated+extracellular+acidification+and+bone+resorption:+evidence+for+a+low+pH+in+resorbing+lacunae+and+localization+of+a+100-kD+lysosomal+membrane+protein+at+the+osteoclast+ruffled+border.">Cell-mediated extracellular acidification and bone resorption: evidence for a low pH in resorbing lacunae and localization of a 100-kD lysosomal membrane protein at the osteoclast ruffled border.</a> Journal of Cell Biology. 101 (6), 2210-2222 (1985).</li><li>Blair, H. C., Teitelbaum, S. L., Ghiselli, R., Gluck, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclastic+bone+resorption+by+a+polarized+vacuolar+proton+pump.">Osteoclastic bone resorption by a polarized vacuolar proton pump.</a> Science. 245 (4920), 855-857 (1989).</li><li>Zhu, 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=Osteoclast-mediated+bone+resorption+is+controlled+by+a+compensatory+network+of+secreted+and+membrane-tethered+metalloproteinases.">Osteoclast-mediated bone resorption is controlled by a compensatory network of secreted and membrane-tethered metalloproteinases.</a> Science Translational Medicine. 12 (529), 6143 (2020).</li><li>Gowen, 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=Cathepsin+K+knockout+mice+develop+osteopetrosis+due+to+a+deficit+in+matrix+degradation+but+not+demineralization.">Cathepsin K knockout mice develop osteopetrosis due to a deficit in matrix degradation but not demineralization.</a> The Journal of Bone and Mineral Research. 14 (10), 1654-1663 (1999).</li><li>Abu-Amer, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-4+abrogates+osteoclastogenesis+through+STAT6-dependent+inhibition+of+NF-kappaB.">IL-4 abrogates osteoclastogenesis through STAT6-dependent inhibition of NF-kappaB.</a> Journal of Clinical Investigation. 107 (11), 1375-1385 (2001).</li><li>Patntirapong, S., Habibovic, P., Hauschka, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+soluble+cobalt+and+cobalt+incorporated+into+calcium+phosphate+layers+on+osteoclast+differentiation+and+activation.">Effects of soluble cobalt and cobalt incorporated into calcium phosphate layers on osteoclast differentiation and activation.</a> Biomaterials. 30 (4), 548-555 (2009).</li><li>Sorensen, M. G., 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=Characterization+of+osteoclasts+derived+from+CD14++monocytes+isolated+from+peripheral+blood.">Characterization of osteoclasts derived from CD14+ monocytes isolated from peripheral blood.</a> The Journal of Bone and Mineral Metabolism. 25 (1), 36-45 (2007).</li><li>Kumar, A., 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=Synergistic+effect+of+biphasic+calcium+phosphate+and+platelet-rich+fibrin+attenuate+markers+for+inflammation+and+osteoclast+differentiation+by+suppressing+NF-kappaB/MAPK+signaling+pathway+in+chronic+periodontitis.">Synergistic effect of biphasic calcium phosphate and platelet-rich fibrin attenuate markers for inflammation and osteoclast differentiation by suppressing NF-kappaB/MAPK signaling pathway in chronic periodontitis.</a> Molecules. 26 (21), 6578 (2021).</li><li>Henriksen, K., Karsdal, M. A., Taylor, A., Tosh, D., Coxon, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+osteoclasts+from+peripheral+blood.">Generation of human osteoclasts from peripheral blood.</a> Methods in Molecular Biology. 816, 159-175 (2012).</li><li>Maria, S. 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=Reproducible+quantification+of+osteoclastic+activity:+characterization+of+a+biomimetic+calcium+phosphate+assay.">Reproducible quantification of osteoclastic activity: characterization of a biomimetic calcium phosphate assay.</a> Journal of Biomedical Materials Research Part B: Applied Biomaterials. 102 (5), 903-912 (2014).</li><li>Kong, L., Smith, W., Hao, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+RAW264.7+for+osteoclastogensis+study:+Phenotype+and+stimuli.">Overview of RAW264.7 for osteoclastogensis study: Phenotype and stimuli.</a> Journal of Cellular and Molecular Medicine. 23 (5), 3077-3087 (2019).</li><li>Li, P., 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=Systemic+tumor+necrosis+factor+alpha+mediates+an+increase+in+peripheral+CD11bhigh+osteoclast+precursors+in+tumor+necrosis+factor+alpha-transgenic+mice.">Systemic tumor necrosis factor alpha mediates an increase in peripheral CD11bhigh osteoclast precursors in tumor necrosis factor alpha-transgenic mice.</a> Arthritis &amp; Rheumatology. 50 (1), 265-276 (2004).</li><li>Arai, F., 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=Commitment+and+differentiation+of+osteoclast+precursor+cells+by+the+sequential+expression+of+c-Fms+and+receptor+activator+of+nuclear+factor+kappaB+(RANK)+receptors.">Commitment and differentiation of osteoclast precursor cells by the sequential expression of c-Fms and receptor activator of nuclear factor kappaB (RANK) receptors.</a> Journal of Experimental Medicine. 190 (12), 1741-1754 (1999).</li><li>Xing, 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=NF-kappaB+p50+and+p52+expression+is+not+required+for+RANK-expressing+osteoclast+progenitor+formation+but+is+essential+for+RANK-+and+cytokine-mediated+osteoclastogenesis.">NF-kappaB p50 and p52 expression is not required for RANK-expressing osteoclast progenitor formation but is essential for RANK- and cytokine-mediated osteoclastogenesis.</a> The Journal of Bone and Mineral Research. 17 (7), 1200-1210 (2002).</li><li>Miyamoto, T., 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=Bifurcation+of+osteoclasts+and+dendritic+cells+from+common+progenitors.">Bifurcation of osteoclasts and dendritic cells from common progenitors.</a> Blood. 98 (8), 2544-2554 (2001).</li></ol>

The authors have nothing to disclose.

Here we describe a simple and reliable method for an osteoclastic resorption assay using OCs derived and expanded in vitro from PBMCs. The used CaP coated cell culture plates can be easily prepared and visualized using lab-available materials. In addition to unsorted PBMCs adopted in this protocol, OCs generated from murine monocytic cells21 and bone marrow macrophage cells17 have also been cultured on similar synthetic substrates for pit assay, thus these cell sou...

<a href="https://app.jove.com/t/6601">This corrects</a> the article <a href="https://dx.doi.org/10.3791/64016">10.3791/64016</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64016">A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro</a>. The Protocol section was&#160;updated.

Erratum: A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

Osteoclasts (OCs) are tissue-specific macrophages derived from hematopoietic stem cells (HSCs), playing a pivotal role in bone remodeling together with osteoblasts1. Sex hormone-induced, immunological, and malignant bone disorders that destroy bone systemically or locally are due to excess osteoclastic activity, including menopause-related osteoporosis2, rheumatoid arthritis3, periodontal disease4, myeloma bone disease5, and osteolytic bone metastasis6. In contrast, defects in OC formation and function can also cause osteopetrosis7. HSCs undergo differentiation into OC progenitors under macrophage colony-stimulating factor (M-CSF, gene symbol ACP5) stimulation. In the presence of both M-CSF and receptor activator of NF-&#954;B ligand (RANKL, gene symbol TNFSF11), OC progenitors differentiate further into mononuclear OCs and subsequently fuse to become multinucleated OCs8,9,10. Both cytokines M-CSF and RANKL are indispensable and sufficient for induction of osteoclastic markers such as calcitonin receptor (CT), receptor activator of nuclear factor &#954; B (RANK), proton pump V-ATPase, chloride channel 7 alpha subunit (CIC-7), integrin &#946;3, tartrate-resistant acid phosphatase (TRAP, gene symbol ACP5), lysosomal cysteine protease cathepsin K (CTSK), and matrix metallopeptidase 9 (MMP9). Activated OCs form a sealing zone on the bone surface through the formation of an actin ring with a ruffled border11,12. Within the sealing zone, OCs mediate resorption through secreting protons via the proton pump V-ATPase12,13, MMP914, and CTSK15, leading to the formation of lacunae.
For in vitro experiments, OC progenitors can be obtained by expansion of bone marrow macrophages from mice&#39;s femur and tibia16,17, as well as by isolation of human peripheral blood mononuclear cells (PBMCs) from blood samples and buffy coats18,19,20, or by differentiation of the immortalized murine monocytic cells RAW 264.721,22.
In the present protocol, we describe an osteoclastic resorption assay in CaP coated cell culture plates using OCs derived from primary PBMCs. The CaP coated cell culture plates method used here are adopted and refined from the method described previously by Patntirapong et al.17 and Maria et al.21. To obtain OC precursors, PBMCs are isolated by density gradient centrifugation and expanded as described previously20.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AgNO3</td>
			<td>SERVA Electrophoresis GmbH</td>
			<td>35110</td>
			<td>Silver nitrate</td>
		</tr>
		<tr>
			<td>a-MEM</td>
			<td>Gibco</td>
			<td>32561-029</td>
			<td>MEM alpha, GlutaMAX, no nucleosides</td>
		</tr>
		<tr>
			<td>amphotericin B</td>
			<td>Biochrom</td>
			<td>03-028-1B</td>
			<td>Amphotericin B Solution</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>21097-50G</td>
			<td>Calcium chloride Dihydrate</td>
		</tr>
		<tr>
			<td>Calcein</td>
			<td>Sigma-Aldrich</td>
			<td>C0875</td>
			<td>Calcein</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F7524</td>
			<td>fetal bovine serum</td>
		</tr>
		<tr>
			<td>Ficoll</td>
			<td>Cytiva</td>
			<td>17144002</td>
			<td>Ficoll Paque Plus</td>
		</tr>
		<tr>
			<td>Fixation buffer</td>
			<td>Biolegend</td>
			<td>420801</td>
			<td>Paraformaldehyde</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Merk</td>
			<td>1.09057.1000</td>
			<td>Hydrochloric acid</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Promokine</td>
			<td>PK-CA707-40046</td>
			<td>Hoechst 33342</td>
		</tr>
		<tr>
			<td>M-CSF</td>
			<td>PeproTech</td>
			<td>300-25</td>
			<td>Recombinant Human M-CSF</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>7791-18-6</td>
			<td>Magnesium chloride</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>AppliChem GmbH</td>
			<td>A2943,0250</td>
			<td>di- Sodium hydrogen phosphate anhydrous</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Merk</td>
			<td>S7653-250G</td>
			<td>Sodium chloride</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Merk</td>
			<td>K15322429</td>
			<td>Bicarbonate of Soda</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Lonza</td>
			<td>17-512F</td>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (1X), DBPS without Calcium and Magnesium</td>
		</tr>
		<tr>
			<td>Pen-Strep</td>
			<td>Lonza</td>
			<td>DE17-602E</td>
			<td>Penicillin-Streptomycin Mixture</td>
		</tr>
		<tr>
			<td>Phalloidin-Alexa Fluor 546</td>
			<td>Invitrogen</td>
			<td>A22283</td>
			<td>Alexa Fluor 546 Phalloidin</td>
		</tr>
		<tr>
			<td>RANKL</td>
			<td>PeproTech</td>
			<td>310-01</td>
			<td>Recombinant Human sRANK Ligand (E.coli derived)</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma-Aldrich</td>
			<td>93362</td>
			<td>Tris(hydroxymethyl)aminomethan</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Alkyl Phenyl Polyethylene Glycol</td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>Gibco</td>
			<td>12605010</td>
			<td>Recombinant cell-dissociation enzymes</td>
		</tr>
	</tbody>
</table>

a simple pit assay protocol to visualize and quantify osteoclastic resorption in vitro

Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases (e.g., osteoporosis and bone cancer) and are therefore important objects of research. In vitro, their activity can be analyzed by the formation of resorption pits. In this protocol, we describe a simple pit assay method using calcium phosphate (CaP) coated cell culture plates, which can be easily visualized and quantified. Osteoclast precursors derived from human peripheral blood mononuclear cells (PBMCs) were cultured on the coated plates in the presence of osteoclastogenic stimuli. After 9 days of incubation, osteoclasts were fixed and stained for fluorescence imaging while the CaP coating was counterstained by calcein. To quantify the resorbed area, the CaP coating on plates was stained with 5% AgNO3 and visualized by brightfield imaging. The resorption pit area was quantified using ImageJ.

The calcium phosphate coating on the bottom of cell culture plates was performed in two coating steps comprising a 3-day pre-calcification and a 1-day calcification step. As shown in Figure 1, uniformly distributed calcium phosphate was obtained on the bottom of the 96-well plates. The coating adhered very well to the bottom after the performed washing steps.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/6401...

Watch this Scientific Journal Video about A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro at JoVE.com

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

Here, we present a simple and effective assay procedure for resorption pit assays using calcium phosphate coated cell culture plates.

A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro

Mature osteoclasts are multinucleated cells that can degrade bone through the secretion of acids and enzymes. They play a crucial role in various diseases ...

Our protocol describes a simple and reliable method for an osteoclastic resorption assay. The use of calcium phosphate coated cell culture plates can be easily prepared and visualized using lab available materials. In contrast to opaque bone or dentin slices, the calcium phosphate coating allows visualization of resorption pits and cells at the same time.In this protocol, we use PBMCs for osteoclast differentiation. However, it can also be applied to other cell types like bone marrow derived monocytes or Raw 264.7 cells. First take a flat bottom 96 well cell culture plate and pipette 300 microliters of previously prepared calcium phosphate solution into each well.Then, cover the plate with the lid and incubate the plate at 37 degrees Celsius for three days. After three days, aspirate the pre-calcification solution from the pre-calcified 96 well cell culture plates and add 300 microliters of calcium phosphate solution to each well. Cover the plates with a lid and incubate at 37 degrees Celsius for one day.Then invert the plate to pour out the solution and thoroughly wash the plate thrice with deionized water. Immediately dry the plate using a hair dryer or compressed carbon dioxide or nitrogen gas to maintain a uniform surface. Now, irradiate the coated plate on a clean bench with ultraviolet rays for one hour.Use the coated plate immediately or seal the plate with Parafilm and store it at room temperature. Dilute 15 milliliters of fresh blood with an equal volume of PBS and mix them by inverting or pipetting several times. Then take 15 milliliters of density gradient solution in a 50 milliliter conical tube, tilt the tube, and carefully layer 30 milliliters of diluted blood sample on the 15 milliliters of density gradient solution.Later, centrifuge the tube in a swinging bucket rotor without break at 810 times G for 20 minutes at 20 degrees Celsius. Then aspirate the upper layer leaving the mononuclear cell layer undisturbed at the interface. Carefully transfer the mononuclear cell layer to a new 50 milliliter conical tube.Fill the tube with PBS, mix, and centrifuge at 300 times G for 10 minutes at 20 degrees Celsius. Re-suspend PBMCs in a complete alpha-MEM containing 20 nanograms per milliliters macrophage colony stimulating factor, and seed 2.5 times 10 to the fifth cells per centimeter square PBMCs in a flask. Incubate the calcium phosphate coated plates with 50 microliters of fetal bovine serum at 37 degrees Celsius for one hour.Next, wash the flask twice with PBS to detach the osteoclast precursors from dead or non-adherent cells, then add four milliliters of trypsin per 75 square centimeter flask. After 30 minutes, stop the digestion by adding four milliliters of complete alpha-MEM, then carefully detach the cells using a cell scraper. Transfer the cells into a 50 milliliter tube and count the total number of cells using a counting chamber.Centrifuge the tube at 350 times G for seven minutes to pellet the cells and resusspend the pellet and complete alpha MEM containing M-CSF and RANKL to get one times 10 to the six cells per milliliter. Aspirate the fetal bovine serum from the calcium phosphate coated plate, and pipette 200 microliters of cell suspension per well. After the incubation, wash the cells twice with PBS.Fix the cells with 4%paraformaldehyde for 10 minutes and wash again with PBS. For staining, add permeabilization buffer to the fixed cells and incubate them for five minutes. To stain the actin filaments, incubate the cells with 100 microliters of Alexa Fluor 546 labeled phalloidin solution in PBS for 30 minutes, then aspirate the staining solution and stain the nuclei using 100 microliters of Hoechst for 10 minutes.Stain calcium phosphate coating with 100 microliters of 10 micromolar calcein and PBS for 10 minutes. After washing thrice with PBS, capture images. For Von Kossa staining of calcium phosphate coating add 50 microliters of 5%silver nitrate and deionized water to each well, and incubate the plate under ultraviolet radiation for one hour until the coating on the bottom of the wells has turned brown.The calcium phosphate coating at the bottom of the 96 well plates appeared to be uniform and well adhered. After nine days of culture in M-CSF and RANKL on the calcium phosphate coated plates, osteoclast precursors formed resorption pits and large osteoclasts. The multi-nucleated osteoclasts located in the pits expressed high TRAP levels.The mature osteoclasts showed the presence of three or more nuclei and the distinct actin ring. The green calcium phosphate coating showed the presence of black resorption pits. The pit area fluorescence images was measured using the thresholding tool of image J.The number and size of osteoclasts were calculated using ROI manager and the polygon selection tool of image J.Osteoclasts'precursors grown in the absence of RANKL could not form resorption pits, however M-CSF and RANKL facilitated the formation of osteoclastogenic pits.And the pit area was quantified using image J.Immediately drying the coated plate with airflow helps to make a uniform calcium phosphate coating. Otherwise it's prone to form a thicker coating in the middle of the well.

a-simple-pit-assay-protocol-to-visualize-quantify-osteoclastic

Research

JoVE Journal

Medicine

5.3K Views.  University Hospital Tübingen. Our protocol describes a simple and reliable method for an osteoclastic resorption assay. The use of calcium phosphate coated cell culture plates can be easily prepared and visualized using lab available materials. In contrast to opaque bone or dentin slices, the calcium phosphate coating allows visualization of resorption pits and cells at the same time.In this protocol, we use PBMCs for osteoclast differentiation. However, it can also be applied to other cell types like bone marrow d...