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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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 ...

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JoVE Journal

Medicine

5.6K Views.  University Hospital Tübingen. Here, we present a simple and effective assay procedure for resorption pit assays using calcium phosphate coated cell culture plates.

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

Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. Conventional methods assess neurological deficits and electrophysiology and quantitative sensory testing quantifies functional alterations to detect neuropathy. However, the earliest damage appears to be to the small fibres and yet these tests primarily assess large fibre dysfunction and have a limited ability to demonstrate regeneration and repair. The only techniques which allow a direct examination of unmyelinated nerve fibre damage and repair are sural nerve biopsy with electron microscopy and skin-punch biopsy. However, both are invasive procedures and require lengthy laboratory procedures and considerable expertise. Corneal Confocal microscopy is a non-invasive clinical technique which provides in-vivo imaging of corneal nerve fibres. We have demonstrated early nerve damage, which precedes loss of intraepidermal nerve fibres in skin biopsies together with stratification of neuropathic severity and repair following pancreas transplantation in diabetic patients. We have also demonstrated nerve damage in idiopathic small fibre neuropathy and Fabry's disease.

Corneal Confocal microscopy is a non-invasive clinical technique which may be used to quantify C fibre damage to diagnose and stratify patients with increasing neuropathic severity.

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. ...

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&amp;#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

A Sensitive Method to Quantify Senescent Cancer Cells

Human cells do not indefinitely proliferate. Upon external and/or intrinsic cues, cells might die or enter a stable cell cycle arrest called senescence. Several cellular mechanisms, such as telomere shortening and abnormal expression of mitogenic oncogenes, have been shown to cause senescence. Senescence is not restricted to normal cells; cancer cells have also been reported to senesce.
	Chemotherapeutical drugs have been shown to induce senescence in cancer cells. However, it remains controversial whether senescence prevents or promotes tumorigenesis. As it might eventually be patient-specific, a rapid and sensitive method to assess senescence in cancer cell will soon be required.
	To this end, the standard &beta;-galactosidase assay, the currently used method, presents major drawbacks: it is time consuming and not sensitive. We propose here a flow cytometry-based assay to study senescence on live cells. This assay offers the advantage of being rapid, sensitive, and can be coupled to the immunolabeling of various cellular markers.

Whether senescence prevents or promotes tumorigenesis remains controversial. Since chemotherapeutical drugs can induce cancer cells to senesce, studying senescence is essential for proposing new therapies. However, the standard and broadly used &beta;-galactosidase assay presents major drawbacks. We propose here a rapid and sensitive flow cytometry-based assay to quantify senescence.

Human cells do not indefinitely proliferate. Upon external and/or intrinsic  cues, cells might die or enter a stable cell cycle arrest called senescence. ...

Parabiosis in Mice: A Detailed Protocol

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of microvasculature at the site of inflammation. Parabiotic partners share their circulating antigens and thus are free of adverse immune reaction. First described by Paul Bert in 18641, the parabiosis surgery was refined by Bunster and Meyer in 1933 to improve animal survival2. In the current protocol, two mice are surgically joined following a modification of the Bunster and Meyer technique. Animals are connected through the elbow and knee joints followed by attachment of the skin allowing firm support that prevents strain on the sutured skin. Herein, we describe in detail the parabiotic joining of a ubiquitous GFP expressing mouse to a wild type (WT) mouse. Two weeks after the procedure, the pair is separated and GFP positive cells can be detected by flow cytometric analysis in the blood circulation of the WT mouse. The blood chimerism allows one to examine the contribution of the circulating cells from one animal in the other.

Parabiotic joining of two organisms leads to the development of a shared circulatory system. In this protocol, we describe the surgical steps to form a parabiotic connection between a wild-type mouse and a constitutive GFP-expressing mouse.

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of ...

Cell-based Assay Protocol for the Prognostic Prediction of Idiopathic Scoliosis Using Cellular Dielectric Spectroscopy

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the prognosis of idiopathic scoliosis in asymptomatic and affected children. The assay consists of the evaluation of the functional status of Gi and Gs proteins in peripheral blood mononuclear cells (PBMCs) by cellular dielectric spectroscopy (CDS), using an automated CDS-based instrument, and the classification of children into three functional groups (FG1, FG2, FG3) with respect to the profile of imbalance between the degree of response to Gi and Gs proteins stimulation. The classification is further confirmed by the differential effect of osteopontin (OPN) on response to Gi stimulation among groups and the severe progression of disease is referenced by FG2. Approximately, a volume of 10 ml of blood is required to extract PBMCs by Ficoll-gradient and cells are then stored in liquid nitrogen. The adequate number of PBMCs to perform the assay is obtained after two days of cell culture. Essentially, cells are first incubated with phytohemmaglutinin (PHA). After 24 hr incubation, medium is replaced by a PHA-free culture medium for an additional 24 hr prior to cell seeding and OPN treatment. Cells are then spectroscopically screened for their responses to somatostatin and isoproterenol, which respectively activate Gi and Gs proteins through their cognate receptors. Both somatostatin and isoproterenol are simultaneously injected with an integrated fluidics system and the cells&#39; responses are monitored for 15 min. The assay can be performed with fresh or frozen PBMCs and the procedure is completed within 4 days.

A structured protocol is presented for a cell-based assay as a functional test to predict the prognosis of idiopathic scoliosis using cellular dielectric spectroscopy (CDS). The assay can be performed with fresh or frozen peripheral blood mononuclear cells (PBMCs) and the procedure is completed within 4 days.

This protocol details the experimental and analytical procedure for a cell-based assay developed in our laboratory as a functional test to predict the ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card,&#160;tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.

The use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations is described.

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be important in the development of future novel therapies. Often&#160;potential new biomarkers do not make it&#160;into the clinic&#160;due to limitations&#160;in their development and&#160;high costs.&#160;However,&#160;targeted proteomics using Multiple Reaction Monitoring Liquid Chromatography-tandem/Mass Spectrometry (MRM LC-MS/MS), specifically using triple quadrupole mass spectrometers, is one method that can be used to rapidly evaluate and validate biomarkers&#160;for clinical translation into diagnostic laboratories. Traditionally, this platform has been used extensively for measurement of small molecules&#160;in clinical laboratories, but it is the potential to analyze proteins, that makes it an attractive alternative to ELISA (Enzyme-Linked Immunosorbent Assay)-based methods. We describe here how targeted proteomics can be used to measure multiplexed markers of dementia, including the detection and quantitation of the known risk factor apolipoprotein E isoform 4 (ApoE4).
In order to make the assay suitable for translation, it is designed to be rapid, simple, highly specific and cost effective. To achieve this, every step in the development of the assay must be optimized for the individual proteins and tissues they are analyzed in. This method describes a typical workflow including various tips and tricks to developing a targeted proteomics MRM LC-MS/MS for translation.
The method development is optimized using custom synthesized versions of tryptic quantotypic peptides, which calibrate&#160;the MS for detection and then spiked into CSF to determine correct identification of the endogenous peptide in the chromatographic separation prior to analysis in the MS. To achieve absolute quantitation, stable isotope-labeled internal standard versions of the peptides with short amino acid sequence tags and containing a trypsin cleavage site, are included in the assay.

We describe a high-throughput, multiplex, and targeted proteomic cerebrospinal fluid (CSF) assay developed with potential for clinical translation. The test can quantitate potential markers and risk factors for neurodegeneration, such as the apolipoprotein E variants (E2, E3 and E4), and measure their allelic expression.

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be ...