Name: methods for analyzing the impacts of natural uranium on in vitro osteoclastogenesis
Uploaded: 2018-01-30T13:00:00
Description: Watch this Scientific Journal Video about Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis at JoVE.com

The authors would like to thank Chantal Cros for helpful technical assistance. 
This research was funded by grants from the &#34;Commissariat &#224; l&#39;Energie Atomique et aux Energies Alternatives&#34; (URANOs - Programme Transversal de Toxicologie du CEA and CPRR CEA-AREVA), and from ANR (Toxicity of URanium: Multi-level approach of biomineralization process in BOne, ANR-16-CE34-0003). This work was also supported by the University of Nice Sophia-Antipolis and the CNRS.

1. Preparation of Uranyl Acetate Solution
<ol>
	<li>To prepare 2 mL of a 100 mM uranyl acetate solution, add 85 mg of uranyl acetate (UO2(OCOCH3)2, 2H2O; M = 424 g.mol-1) in solid state to a 5 mL plastic tube.</li>
	<li>Add 2 mL of distilled water to the plastic tube and fit it with a plastic stopper.</li>
	<li>Shake the tube vigorously until the total dissolution of the solid. Put the solution into the fridge for 24 h. 
	CAUTION: The manipulation of uranyl [U...

<ol>
	<li>Keith, S., Faroon, O., Roney, N., Scinicariello, F., Wilbur, S., Ingerman, 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=Toxicological+profile+for+uranium.">Toxicological profile for uranium.</a> Agency for Toxic Substances and Disease Registry. , (2013).</li><li>Ballou, J. E., Gies, R. A., Case, A. C., Haggard, D. L., Buschbom, R. L., Ryan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deposition+and+early+disposition+of+inhaled+233UO2(NO3)2+and+232UO2(NO3)2+in+the+rat.">Deposition and early disposition of inhaled 233UO2(NO3)2 and 232UO2(NO3)2 in the rat.</a> Health Phys. 51 (6), 755-771 (1986).</li><li>Kathren, R. L., McInroy, J. F., Moore, R. H., Dietert, S. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+osteoclast+and+its+unique+cytoskeleton.">The osteoclast and its unique cytoskeleton.</a> Ann N Y Acad Sci. 1240, 14-17 (2011).</li><li>Nesbitt, S. A., Horton, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trafficking+of+matrix+collagens+through+bone-resorbing+osteoclasts.">Trafficking of matrix collagens through bone-resorbing osteoclasts.</a> Science. 276 (5310), 266-269 (1997).</li><li>Salo, J., Lehenkari, P., Mulari, M., Metsikko, K., Vaananen, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+osteoclast+bone+resorption+products+by+transcytosis.">Removal of osteoclast bone resorption products by transcytosis.</a> Science. 276 (5310), 270-273 (1997).</li><li>Pierrefite-Carle, V., 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=Effect+of+natural+uranium+on+the+UMR-106+osteoblastic+cell+line:+impairment+of+the+autophagic+process+as+an+underlying+mechanism+of+uranium+toxicity.">Effect of natural uranium on the UMR-106 osteoblastic cell line: impairment of the autophagic process as an underlying mechanism of uranium toxicity.</a> Arch. Toxicol. 91 (4), 1903-1914 (2017).</li><li>Ubios, A. M., Guglielmotti, M. B., Steimetz, T., Cabrini, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uranium+inhibits+bone+formation+in+physiologic+alveolar+bone+modeling+and+remodeling.">Uranium inhibits bone formation in physiologic alveolar bone modeling and remodeling.</a> Environ. Res. 54 (1), 17-23 (1991).</li><li>Bozal, C. B., Martinez, A. B., Cabrini, R. L., Ubios, A. M. <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+ethane-1-+hydroxy-1,1-bisphosphonate+(EHBP)+on+endochondral+ossification+lesions+induced+by+a+lethal+oral+dose+of+uranyl+nitrate.">Effect of ethane-1- hydroxy-1,1-bisphosphonate (EHBP) on endochondral ossification lesions induced by a lethal oral dose of uranyl nitrate.</a> Arch. Toxicol. 79 (8), 475-481 (2005).</li><li>Kurttio, 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=Bone+as+a+possible+target+of+chemical+toxicity+of+natural+uranium+in+drinking+water.">Bone as a possible target of chemical toxicity of natural uranium in drinking water.</a> Environ. Health Perspect. 113 (1), 68-72 (2005).</li><li>Collin-Osdoby, P., Osdoby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RANKL-mediated+osteoclast+formation+from+murine+RAW+264.7+cells.">RANKL-mediated osteoclast formation from murine RAW 264.7 cells.</a> Methods Mol. Biol. 816, 187-202 (2012).</li><li>Beranger, G. 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=Differential+binding+of+poly(ADP-Ribose)+polymerase-1+and+JunD/Fra2+accounts+for+RANKL-induced+Tcirg1+gene+expression+during+osteoclastogenesis.">Differential binding of poly(ADP-Ribose) polymerase-1 and JunD/Fra2 accounts for RANKL-induced Tcirg1 gene expression during osteoclastogenesis.</a> J. Bone Miner. Res. 22 (7), 975-983 (2007).</li><li>Mirto, 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=Influence+of+uranium(VI)+speciation+for+the+evaluation+of+in+vitro+uranium+cytotoxicity+on+LLC-PK1+cells.">Influence of uranium(VI) speciation for the evaluation of in vitro uranium cytotoxicity on LLC-PK1 cells.</a> Hum. Exp. Toxicol. 18 (3), 180-187 (1999).</li><li>Carri&#232;re, 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=Influence+of+uranium+speciation+on+normal+rat+kidney+(NRK-52E)+proximal+cell+cytotoxicity.">Influence of uranium speciation on normal rat kidney (NRK-52E) proximal cell cytotoxicity.</a> Chem. Res. Toxicol. 17 (3), 446-452 (2004).</li><li>Milgram, S., Carri&#232;re, M., Malaval, L., Gouget, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+accumulation+and+distribution+of+uranium+and+lead+in+osteoblastic+cells+as+a+function+of+their+speciation.">Cellular accumulation and distribution of uranium and lead in osteoblastic cells as a function of their speciation.</a> Toxicology. 252 (1-3), 26-32 (2008).</li><li>Milgram, S., Carri&#232;re, M., Thiebault, C., Malaval, L., Gouget, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxic+and+phenotypic+effects+of+uranium+and+lead+on+osteoblastic+cells+are+highly+dependent+on+metal+speciation.">Cytotoxic and phenotypic effects of uranium and lead on osteoblastic cells are highly dependent on metal speciation.</a> Toxicology. 250 (1), 62-69 (2008).</li><li>Studzinski, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Growth, Differentiation and Senescence A Practical Approach. , (1999).</li><li>Gritsaenko, 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=Natural+uranium+impairs+the+differentiation+and+the+resorbing+function+of+osteoclasts.">Natural uranium impairs the differentiation and the resorbing function of osteoclasts.</a> Biochim Biophys Acta. 1861 (4), 715-726 (2017).</li><li>Motiur Rahman, 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=Proliferation-coupled+osteoclast+differentiation+by+RANKL:+Cell+density+as+a+determinant+of+osteoclast+formation.">Proliferation-coupled osteoclast differentiation by RANKL: Cell density as a determinant of osteoclast formation.</a> Bone. 81, 392-399 (2015).</li></ol>

As far as we know, this is the first time that a detailed procedure aiming to study the effect of natural uranium on bone resorbing cells is described. This approach will be useful to achieve a better understanding of uranium impact on bone physiology and may provide an interesting new tool for the screening of uranium chelators. Furthermore, we believe that the protocol described here could be applied to study the impact of other heavy metals on osteoclatogenesis.
It is known that uranyl is c...

Uranium is a naturally occurring radioactive element present in soils, air and water; as such, animals and humans are exposed to natural uranium in their diets. In addition to natural sources, uranium originates from anthropogenic activities, which increases its abundance in the environment. Uranium poses both chemical and radiological hazards. However, because natural uranium (which is an isotopic mixture containing 99.27% 238U, 0.72% 235U, and 0.006% 234U) has a low specific activity (25.103 Bq.g-1), its impacts on health are attributed to its chemical toxicity.
Whatever its entry route (inhalation, ingestion, or dermal exposure), most of uranium entering the body is eliminated with feces and only a small portion reaches the systemic circulation. Approximately 67% of uranium in the blood is in turn filtered by kidneys and leaves the body in urine within 24 h1. The remainder is mostly deposited in kidneys and bones, the two main target organs of uranium toxicity2,3,4. Because the skeleton has been identified as the primary site of uranium long-term retention2,3,4,5,6, several studies have been conducted to explore the effect of uranium on bone physiology7.
Bone is a mineralized tissue that is continuously remodeled throughout its lifetime. Bone remodeling is a complex process that depends on specialized cell types and consists mostly of two phases: resorption of the pre-existing old matrix by osteoclasts followed by de novo bone construction by osteoblasts. Osteoclasts are large multinuclear cells resulting from the fusion of precursor cells of hematopoietic origin that migrate to resorption sites where they attach to bone8. Their attachment occurs simultaneously with an extensive reorganization of their cytoskeleton9. This reorganization is required for the establishment of an isolated compartment between the cell and the bone surface into which the osteoclast secretes protons, leading to the dissolution of hydroxyapatite, and proteases involved in the degradation of the organic matrix. The resulting degradation products are endocytosed, transported through the cell to the membrane area opposite to the bone surface and secreted, a process called transcytosis10,11.
Results from in vivo and in vitro studies indicate that uranium inhibits bone formation and alters the number and the activity of osteoblasts7,12. In contrast, the effects of uranium on bone resorption and osteoclasts have been poorly explored. Several in vivo studies have reported an enhancement of bone resorption after administration of uranyl nitrate to mice or rats13,14. Furthermore, an epidemiological investigation suggested that the increase in uranium intake via drinking water tended to be associated with an increase in the serum level of a bone resorption marker in men15. Taken together, these findings led to the conclusion that uranium, which accumulates in bone could promote bone resorption. However, the cellular mechanisms involved in this potential effect of uranium remain an open question. For this reason, we decided to examine the impact of uranium on behavior of resorbing bone cells.
Here, we describe the protocol we have established to characterize and quantify the effects of natural uranium on pre-osteoclasts viability and on osteoclasts differentiation and resorptive activity. The experiments described herein have been done with the RAW 264.7 murine transformed macrophage cell line, which can readily differentiate into osteoclasts when cultured in the presence of the cytokine RANKL for 4 or 5 days, and which is classically used to study osteoclast differentiation and function16. The procedures developed are reliable, give highly reproducible results and are fully applicable to primary osteoclasts. For all these reasons, we believe that this methodology is useful for getting a better understanding of molecular mechanisms involved in uranium toxicity in bone. Moreover, we think that this approach could be adapted as a screening tool for identifying new uranium chelating agents.

<table>
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Lonza</td>
			<td>BE12-604F</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-MEM</td>
			<td>Lonza</td>
			<td>BE12-169F</td>
			<td></td>
		</tr>
		<tr>
			<td>EMEM without phenol red</td>
			<td>Lonza</td>
			<td>12-668E</td>
			<td></td>
		</tr>
		<tr>
			<td>Water for cell culture</td>
			<td>Lonza</td>
			<td>BE17-724F</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin solution</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;L-Glutamine solution</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution 0.4%</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>HyClone fetal bovine serum</td>
			<td>GE Life Sciences</td>
			<td>SH30071.03</td>
			<td></td>
		</tr>
		<tr>
			<td>7.5% sodium bicarbonate aqueous solution</td>
			<td>Sigma-Aldrich</td>
			<td>S8761</td>
			<td></td>
		</tr>
		<tr>
			<td>Acid Phosphatase, Lekocyte (TRAP) kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A</td>
			<td></td>
		</tr>
		<tr>
			<td>Thiazolyl Blue Tetrazolium Bromide (MTT) powder</td>
			<td>Sigma-Aldrich</td>
			<td>M5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D5879</td>
			<td></td>
		</tr>
		<tr>
			<td>Alizarin Red S sodium salt, 1% w/v aq. sol.</td>
			<td>Alfa Aeros</td>
			<td>42746</td>
			<td></td>
		</tr>
		<tr>
			<td>Osteoassay bone resorption plates, 24 well plates</td>
			<td>Corning Life Sciences</td>
			<td>3987</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiwell 24 well plates</td>
			<td>Falcon</td>
			<td>353504</td>
			<td></td>
		</tr>
		<tr>
			<td>Flask 75 cm2</td>
			<td>Falcon</td>
			<td>353133</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Conical Tubes 50 ml</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scrapers 30 cm</td>
			<td>TPP</td>
			<td>90003</td>
			<td></td>
		</tr>
	</tbody>
</table>

methods for analyzing the impacts of natural uranium on in vitro osteoclastogenesis

Uranium has been shown to interfere with bone physiology and it is well established that this metal accumulates in bone. However, little is known about the effect of natural uranium on the behavior of bone cells. In particular, the impact of uranium on osteoclasts, the cells responsible for the resorption of the bone matrix, is not documented. To investigate this issue, we have established a new protocol using uranyl acetate as a source of natural uranium and the murine RAW 264.7 cell line as a model of osteoclast precursors. Herein, we detailed all the assays required to test uranium cytotoxicity on osteoclast precursors and to evaluate its impact on the osteoclastogenesis and on the resorbing function of mature osteoclasts. The conditions we have developed, in particular for the preparation of uranyl-containing culture media and for the seeding of RAW 264.7 cells allow to obtain reliable and highly reproductive results. Moreover, we have optimized the use of software tools to facilitate the analysis of various parameters such as the size of osteoclasts or the percentage of resorbed matrix.

Tartrate-resistant acid phosphatase staining was used to visualize osteoclasts as large purple cells having 3 or more nuclei. Representative images of osteoclasts obtained from RAW 264.7 cells cultured in the presence of RANKL and uranyl ions are shown in Figure 1. Changes in number and size of osteoclasts in response to uranium are easily visible in composite images of whole wells and in enlarged pictures.
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Watch this Scientific Journal Video about Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis at JoVE.com

Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis

Uranium is known to affect bone metabolism. Here, we present a protocol aimed at investigating the effect of natural uranium exposure on the viability, the differentiation, and the function of osteoclasts, the cells in charge of bone resorption.

Methods for Analyzing the Impacts of Natural Uranium on In Vitro Osteoclastogenesis

Uranium has been shown to interfere with bone physiology and it is well established that this metal accumulates in bone. However, little is known about ...

The overall goal of this procedure is to investigate the effects of natural uranium exposure on osteoclast viability, differentiation and function. This method can help answer key questions in the environmental toxicity field about the impact of uranium exposure on the bone physiology and metabolism. The main advantage of this technique is that it provides an interesting new tool for the screening of uranium originators.When the RAW 264.7 cell culture has reached 85 to 90%confluency, use a cell scraper to mechanically lift the cells from the bottom of the cell culture flask and transfer the cells into a sterile 50-milliliter conical tube. Pipette the cell solution a few times to break up the clumps and count the resulting single-cell suspension. Dilute the cells to a one times 10-to-the-fourth cells-per-milliliter concentration in alpha-modified Minimum Essential Medium, supplemented with L-glutamine and 5%fetal bovine serum and add one milliliter of cells per well to a 24-well cell culture plate for a six-hour incubation at 37 degrees Celsius.An important parameter for the reproducibility of the osteoclast differentiation and reabsorption assay is the cell seeding density. Pay particular attention to the cell counting, cellular suspension preparation and its homogeneity before plating the cells. To prepare the urinal salt solution, starting from a sterile cell culture-tested 7.5%sodium bicarbonate aqueous solution, first prepare and cool 100 millimolar bicarbonate in water on ice and add one volume of 100 millimolar urinal acetate solution, drop by drop, into nine volumes of the ice-cold bicarbonate solution with gentle shaking.Allow the solution to equilibrate at room temperature. After three hours, dilute the appropriate volume of urinal solution, drop by drop, into differentiation medium containing 50 nanograms per milliliter of RANKL until the desired urinal working concentration is achieved and allow another three hours of equilibration at room temperature. The long incubation steps after each dilution of the urinal salt are essential for stabilization of the urinal complexes in the solutions.At the end of the second equilibration period, gently replace the medium in each well with one milliliter of urinal treated or control medium as appropriate and return the cells to the incubator. Multinucleated osteoclasts should appear between days three and four of culture. To assess the size and morphology of the newly differentiated osteoclasts, stain the cells with tartrate-resistant acid phosphatase, according to the manufacturer's instructions and image the whole surface of each well at a resolution sufficient for distinguishing cell nuclei.When all of the wells have been imaged, open an image of one well with open-source ImageJ software and verify the scale units. Then check the Global box in the popup window to apply the new scale units to all of the later images. Under the Analyze menu, select Set Measurements and check the Area and Display label boxes.Uncheck all of the other boxes. Under the Analyze menu, open Tools then the Region of Interest Manager and select the rectangular selection tool in the main ImageJ window. Outline a region of interest of about 1, 000-by-1, 000 micrometers and click the Add button to add the selection to the Region of Interest Manager.Then click More, Save to save the region. To create a copy of one of the five regions to be analyzed, select the region of interest in the Region of Interest Manager and right-click inside the selected area to duplicate the region. Check the Show All and Labels boxes and select the polygon selection tool to manually outline one of the osteoclasts in the duplicated region of interest.To add this outline to the Region of Interest Manager, click Add in the Region of Interest Manager window. When all of the osteoclasts have been added, select all of the outlines and click Measure to measure their surfaces. Then transfer the measurements that appear in the results window to a spreadsheet.To assess the function of urinal treated osteoclasts, one day before adding the differentiation medium, plate RAW cells from an 85 to 90%confluent cell culture on to a 24-well osteo assay plate in growth medium, as just demonstrated. The next day, gently replace the supernatant with differentiation urinal containing medium and allow the RAW cells to differentiate into osteoclasts, as just demonstrated. On day five of osteoclastogenesis, replace the urinal-containing medium with 10%bleach solution to remove the cells from the bone mimetic surface.After five minutes at room temperature, wash the wells two times with deionized water and let them air dry. Next, stain the calcium salts in the non-resorbed areas of each well with a 1%Alizarin Red S sodium salt solution. After 10 to 15 seconds, remove the Alizarin solution and gently wash the wells three to four times with water.To convert an RGB image of the Alizarin-stained wells to a grayscale, 8-bit format, under the Image menu, select Type and 8-bit. Then, using the oval selection tool, outline the entire surface of the well to be analyzed for resorption and save the region of interest, as just demonstrated. To facilitate threshold selection, select Edit and Clear Outside to clear all of the features outside of the defined region of interest.Duplicate the region of interest as just demonstrated and select Adjust Threshold under the Image menu, adjusting the slide bar to select an appropriate threshold. All of the resorbed areas should be above the threshold and appear white, while all of the non-resorbed areas should be below the threshold and appear red. To finalize the threshold selection, click Apply in the Threshold window and save the final binary image in the appropriate format of interest.To define the measurements to be taken within the region of interest, under the Analyze menu, select Set Measurements and check Area, Area Fraction and Limit to Threshold boxes and uncheck all of the other boxes. Then to obtain the fraction of resorbed area directly within the results window, invert the duplicated binary image by selecting Invert under the Edit menu and taking care that the main region of interest is selected on the final binary image and measure the resorbed area fraction. The changes in number and size of newly differentiated osteoclasts in response to uranium are easily visible in composite images of whole wells, as well as in enlarged pictures of select regions of interest.The size of the osteoclasts grown under different culture conditions can be analyzed as just demonstrated by outlining all of the osteoclasts within each region and measuring the area of each outlined cell. To investigate the impact of uranium on osteoclast resorption activity, RAW 264.7 cells are plated and differentiated on osteo mimetic surface plates. At the end of the assay, the osteoclasts are removed and the non-resorbed areas are stained and imaged as demonstrated.The images are then converted to 8-bit grayscale and thresholded to facilitate calculation of the resorbed areas. This procedure can also be applied to the study of the impact of other heavy metals on osteoclastogenesis.

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Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a ...

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Methods for the Study of Regeneration in Stentor

Methods for the Study of Regeneration in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the ...

Methods to Test Endocrine Disruption in Drosophila melanogaster

Methods to Test Endocrine Disruption in Drosophila melanogaster

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...