Name: A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro (Video) | JoVE Journal
Uploaded: 2022-06-16T13:40:00.000+00:00
Duration: 7 min 3 s
Description: Learn a simple pit assay protocol for osteoclastic resorption using calcium phosphate coated plates and PBMCs. This method enables simultaneous visualization of resorption pits and cells for bone biology research.

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5 minutes

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 mM Tris buffer.</li>
		<li>Weigh out 0.368 g of CaCl2&#183;2H2O, 8.0 g of NaCl, and 0.305 g of MgCl2&#183;6H2O and dissolve in Tris buffer one by one.</li>
		<li>Adjust pH to 7.4 using 1 M HCl. Store at room temperature.</li>
	</ol>
	</li>
	<li>Preparation of phosphate stock solution (11.1 mM Na2HPO4, 42 mM NaHCO3 in Tris buffer)
	<ol>
		<li>Set up a glass beaker on a magnetic stirrer and add 100 mL of 50 mM Tris buffer.</li>
		<li>Weigh out 0.158 g of Na2HPO4&#160;and 0.353 g of NaHCO3 and dissolve in Tris buffer one by one.</li>
		<li>Adjust pH to 7.4 using 1 M HCl. Store at room temperature.</li>
	</ol>
	</li>
	<li>Pre-calcification of 96-well cell culture plates
	<ol>
		<li>Prepare 30 mL of working solution by mixing 15 mL of 50 mM Tris buffer, 7.5 mL of calcium stock (step 1.1.4) solution, and 7.5 mL of phosphate stock solution (step 1.2.3). Filter the solution with a 0.2 &#181;m filter.</li>
		<li>Prepare a 96-well cell culture plate with flat bottom. Pipette 300 &#181;L of calcium phosphate solution (step 1.3.1.) to each well. Cover the plate with a lid and incubate the plate at 37 &#176;C for 3 days.</li>
	</ol>
	</li>
	<li>Preparation of calcium phosphate solution (2.25 mM Na2HPO4, 4 mM CaCl2&#183;2H2O, 0.14 M NaCl, 50 mM Tris base in ddH2O)
	<ol>
		<li>Add 2 mL of 1 M HCl to 40 mL of deionized water in a beaker with a magnetic stirring bead and dissolve 0.016 g of Na2HPO4, 0.0295 g of CaCl2&#183;2H2O, 0.409 g of NaCl, and 0.303 g of Tris base one by one.</li>
		<li>Adjust pH to 7.4 using 1 M HCl. Add deionized water to fill the volume to 50 mL. Filter the solution with a 0.2 &#181;m filter.</li>
	</ol>
	</li>
	<li>Calcification of 96-well cell culture plates
	<ol>
		<li>Aspirate pre-calcification solution from the pre-calcified 96-well cell culture plates and add 300 &#181;L of calcium phosphate solution (step 1.4.2) to each well. Cover the plates with a lid and incubate at 37 &#176;C for 1 day.</li>
		<li>Turn the plate over to pour out the solution and wash the plate three times with deionized water thoroughly. Dry the plate immediately using a hair dryer or compressed CO2 or N2 gas to maintain a uniform surface.</li>
		<li>Sterilize the coated plate by UV radiation in a clean bench for 1 h. Use the coated plate immediately or seal the plate with parafilm and store it at room temperature.</li>
	</ol>
	</li>
</ol>
2. Isolation of PBMCs from human peripheral blood
<ol>
	<li>Draw 15 mL of blood from the vein after obtaining written informed consent from the blood donor (ethical vote: 287/2020B02).</li>
	<li>Dilute 15 mL of fresh blood with an equal volume of PBS and mix by inverting the tube several times or by drawing the mixture in and out of a pipette.</li>
	<li>Prepare a 50 mL conical tube containing 15 mL of density gradient solution (e.g., Ficoll, Table of Materials) per tube. Tilt the tube and carefully layer 30 mL of diluted blood sample onto the 15 mL of density gradient solution (diluted blood sample: density gradient solution, 1:0.5-1 ratio). 
	NOTE: When overlaying the sample, pay attention not to mix the density gradient solution with the diluted blood sample.</li>
	<li>Centrifuge at 810 x g for 20 min at 20 &#176;C in a swinging-bucket rotor without brake.</li>
	<li>Aspirate the upper layer leaving the mononuclear cell layer (lymphocytes, monocytes, and thrombocytes) undisturbed at the interphase.</li>
	<li>Carefully transfer the mononuclear cell layer to a new 50 mL conical tube.</li>
	<li>Fill the tube with PBS, mix, and centrifuge at 300 x g for 10 min at 20 &#176;C (brake can be used from this stage onwards).</li>
	<li>Carefully remove supernatant completely. Resuspend the cell pellet in 50 mL of PBS and centrifuge at 300 x g for 10 min at 20 &#176;C. Carefully remove the supernatant completely.</li>
	<li>For removal of platelets, resuspend the cell pellet in 50 mL of PBS and centrifuge at 200 x g for 10 min at 20 &#176;C. Carefully remove the supernatant completely.</li>
	<li>Transfer resuspended cells to cell culture flasks for expansion of OC progenitors.</li>
</ol>
3. Expansion of OC progenitors
<ol>
	<li>Resuspend PBMCs in complete &#945;-MEM (10% FBS, 1% Pen/Strep, 1% amphotericin B) containing 20 ng/mL M-CSF. Seed PBMCs at a density of 2.5 x 105 cells/cm2. Usually, cells isolated from 15-20 mL fresh blood can be seeded in one 75 cm2 flask (1.5-2 x 107 cells).</li>
	<li>Feed the cells with fresh complete &#945;-MEM containing 20 ng/mL M-CSF every third day until the attached cells reach a desired confluency. Typical yields are 1.5-2 x 106 cells/flask when cells are 95% confluent. Usually, the expansion period lasts 6 days. 
	&#8203;NOTE: The osteoclastogenic potential of the precursors can decrease with prolonged cultivation time.</li>
</ol>
4. Induction of osteoclastogenesis in CaP coated plates
<ol>
	<li>To facilitate cell adhesion, incubate the CaP coated plates with 50 &#181;L of FBS for 1 h in a 37 &#176;C incubator.</li>
	<li>To detach the OC precursors, wash the flask with PBS twice to remove dead or non-adherent cells. Add 4 mL of trypsin (Table of Materials) per 75 cm2 flask, for 30 min.
	<ol>
		<li>Add 4 mL of complete &#945;-MEM to stop the digestion, carefully detach the cells using a cell scraper and transfer the cells into a 50 mL tube.</li>
		<li>Determine the total cell number using a Neubauer chamber or similar.</li>
	</ol>
	</li>
	<li>Pellet the cells by centrifugation for 7 min at 350 x g. Resuspend the pellet in sufficient complete &#945;-MEM containing 20 ng/mL M-CSF and 20 ng/mL RANKL to get a concentration of 1 x 106 cells/mL.</li>
	<li>Aspirate FBS solution from the CaP coated 96-well plate and pipette 200 &#181;L of cell suspension per well (2 x 105 cells/well).</li>
	<li>Incubate the OC precursors for the desired time period or with the desired reagents relevant to the experimental design. Usually, high numbers of large and multinucleated OCs can be observed after 6 days and when resorption pits are forming. Feed cells with fresh complete &#945;-MEM medium containing 20 ng/mL M-CSF and 20 ng/mL RANKL every third day.</li>
	<li>At the end of incubation, wash cells with PBS twice, fix with 4% paraformaldehyde for 10 min, and wash with PBS again.</li>
	<li>Use the fixed OCs directly for fluorescence staining or store at 4 &#176;C.</li>
</ol>
5. Fluorescence staining of OCs and CaP coating
<ol>
	<li>Incubate the fixed cells with permeabilization buffer (0.1% Triton in PBS) for 5 min.</li>
	<li>Stain actin filaments with 100 &#181;L of AlexaFluor 546 labeled phalloidin solution in PBS for 30 min and aspirate the staining solution. Add 100 &#181;L of Hoechst 33342 staining solution (10 &#181;g/mL in PBS) for 10 min to stain nuclei. DAPI can also be used.</li>
	<li>Stain CaP coating with 100 &#181;L of 10 &#181;M calcein in PBS for 10 min. Wash three times with PBS and take images.</li>
	<li>After fluorescence imaging the same plates can be used to quantify resorption pit area by Von Kossa staining. To do so, wash the plates with deionized water twice.</li>
</ol>
6. Quantification of total resorption pit area
<ol>
	<li>To stain CaP coating with Von Kossa staining, incubate with 50 &#181;L of 5% AgNO3 in deionized water per well under UV radiation for 1 h, until the coating on the bottom of the wells has turned brown.</li>
	<li>Wash the plate three times with deionized water and take brightfield images. An objective with little magnification, such as a 1.25x objective, can be used to capture the whole well in one image. This facilitates subsequent image analysis. Alternative methods to capture the whole area of the well can be applied.</li>
	<li>Open an image file with ImageJ: File | Open | Image | Type | 8-bit. Verify the scale unit in the lower right corner of the image using Straight Line | Analyze | Set Scale. Enter the length in the &#34;known distance&#34; blanket, enter new scale unit in Unit of length, and check the box Global.</li>
	<li>List measurement parameters to be analyzed: Analyze | Set Measurements. In the Set Measurements window, check the Area and Limit to threshold boxes.</li>
	<li>Measure the area of pits: Image | Adjust | Threshold. In the Threshold window, check box Dark background and click Auto. The area of pits turns red. Close the Threshold window and select Analyze | Measure. Save the result file: File | Save As.</li>
</ol>
7. Quantification of OC number and size, and normalized resorption pit area
<ol>
	<li>Open a fluorescence image of osteoclasts with ImageJ and verify the scale unit (see step 6.3).</li>
	<li>List measurement parameters to be analyzed: Analyze | Set Measurements. In the Set Measurements window, check the Area box.</li>
	<li>Outline the OCs using ROI Manager and Polygon selections: Analyze | Tools | ROI Manager. Click Polygon selections in toolset, outline one OC (cells with actin ring and &#8805; three nuclei) and click Add [t] in ROI Manager window. Repeat outlining until all OCs are included.</li>
	<li>Measure number and size of OCs: Select all items in ROI Manager and click Measure. Save the result file: File | Save As (Figure 3E).</li>
	<li>Measure the pit area of fluorescence images. Open the fluorescent image of coating correlated with the image in step 7.3 and verify the scale unit (see step 6.3).
	<ol>
		<li>List measurement parameters to be analyzed (see step 6.4). Select Image | Adjust | Threshold. In the Threshold window, uncheck all boxes and click Auto. The area of the pits turns red. Close the Threshold window and select Analyze | Measure. Save the result file.</li>
	</ol>
	</li>
	<li>Calculate normalized pit area by OC number.</li>
</ol>

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</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><tbody><tr><td>AgNO&lt;sub&gt;3&lt;/sub&gt;</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>CaCl&lt;sub&gt;2&lt;/sub&gt;</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>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>7791-18-6</td><td>Magnesium chloride</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</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>NaHCO&lt;sub&gt;3&lt;/sub&gt;</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&#039;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

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="Microstructure analysis, grain boundary visualization...

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

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

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Research

JoVE Journal

Medicine

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Learn a simple pit assay protocol for osteoclastic resorption using calcium phosphate coated plates and PBMCs. This method enables simultaneous visualization of resorption pits and cells for bone biology research.

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

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