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(VI)] presents some chemical and radiological hazards. All the samples should be prepared and characterized with ad hoc equipment in specific laboratories. All U(VI)-containing liquids should be collected in specifically assigned waste containers and disposed of as toxic waste. Dry waste (pipets, tubes, etc.) could be washed with 0.1 N HNO3 solution, which will solubilize and remove U(VI).</li>
	<li>Verify the pH of the solution using a microelectrode and a pH-meter. 
	NOTE: The microelectrode should be previously calibrated using commercial buffer solutions (pH= 4 and pH = 7).</li>
</ol>
2. RAW 264.7 Cell Culture
<ol>
	<li>Maintenance of the cells.
	<ol>
		<li>Cultivate RAW 264.7 cells (ATCC, TIB-71) in 75 cm2 flasks in the following growth medium: Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 5% Fetal Bovine Serum and antibiotics: 100 IU/mL penicillin, 100 &#181;g/mL streptomycin. Maintain cells in incubator at 37 &#176;C, 5% CO2.</li>
		<li>Change medium every 2 to 3 days. When the cells are 85-90% confluent, mechanically remove them from the culture flask using a cell scraper (as recommended by the ATCC) and split them at a ratio of 1:6. 
		NOTE:Only cells between passages 3 to 10 are used for differentiation experiments.</li>
	</ol>
	</li>
	<li>Preparation of the cells for experiments
	<ol>
		<li>Dislodge cells from the flask substrate as described above and transfer them to a sterile 50 mL tube. Mix cells by pipetting up and down to break up clumps. Count them with a hemocytometer. Expect 35-40 million cells per 75 cm2 flask.</li>
		<li>Calculate volume of cells suspension necessary for each experiment as follows: 
		Number of experimental conditions x 3 (triplicates) x 1 mL/well + 3-4 mL (pipetting losses). 
		NOTE: Seeding density for all assays described in this protocol is 5,000 cell/cm2. Therefore, for a 24-well plate, it means 10,000 cells per well in 1 mL of medium, and cell suspension density is thus 10,000 cells/mL.</li>
		<li>Prepare necessary volume of 10,000 cells/mL cell suspension and seed the cells into 24-well plates. For cytotoxicity assays, use normal growth medium. For differentiation and resorption assays, use alpha modified Minimum Essential Medium (&#945;MEM) supplemented with 2 mM L-Glutamine, 5% FBS and 50 ng/mL of GST-RANKL17. 
		NOTE: The GST-RANKL protein produced in-house can be replaced by any commercially available RANKL cytokine. When working with a new RANKL batch, it may be useful to test its effectiveness in inducing osteoclastogenesis by performing a dose response experiment with RANKL concentration ranging from 20 to 150 ng/mL.</li>
	</ol>
	</li>
</ol>
3. Dilution of the 100 mM Uranyl Acetate Solution in Culture Medium
NOTE: Uranyl ions [U(VI)] in culture medium form multiple complexes with other medium components that could modify its cellular toxicity18,19,20,21. For this reason, uranyl-containing media should be prepared extemporaneously without omitting or shortening equilibration steps.
<ol>
	<li>Chose uranyl exposure concentrations and calculate volumes of culture media required for the experiment (taking into account that each condition is performed in triplicate).</li>
	<li>Starting from the sterile cell culture tested 7.5% sodium bicarbonate aqueous solution ([HCO3-] = 893 mM), prepare a 100 mM HCO3- solution in water and cool it on ice.</li>
	<li>Add drop-by-drop 1 volume of uranyl acetate 100 mM stock solution to 9 volumes of ice-cold 100 mM HCO3- solution, gently shaking the mixing tube. This operation will dilute uranyl acetate stock solution ([UO22+] = 100 mM, pH 4) to 10 mM and bring the pH to around 7.</li>
	<li>Add 1 volume of sterile water to 9 volumes of ice-cold 100 mM HCO3- solution to reach 90 mM HCO3- (equal to HCO3- concentration in the 10 mM uranyl solution), which will serve to prepare the control medium.</li>
	<li>Allow prepared solutions to stand for 3 h at room temperature (RT) for equilibration.</li>
	<li>In the meantime, prepare the basic exposure medium: &#945;MEM with 2 mM L-Glutamine and 5% FBS. 
	NOTE: For differentiation and resorption assays, add 50 ng/mL of GST-RANKL to the basic exposure medium.</li>
	<li>After 3 h, dilute appropriate amounts of the 10 mM uranyl solution drop by drop in the exposure medium so as to achieve the desired uranyl working concentrations. Prepare simultaneously the control medium by adding the amount of bicarbonate used in the most concentrated uranyl-treated condition, to the exposure medium.</li>
	<li>Allow prepared media to stand for 3 h at RT for equilibration.</li>
	<li>Remove growth media from the wells and replace it with the control and the uranyl-containing media.</li>
</ol>
4. Analysis of RAW 264.7 Precursors Viability in the Presence of U(VI) (MTT Cytotoxic Assays)
CAUTION: Both MTT and DMSO could cause skin and eye irritation. Wear gloves and eye protection when handling them. Collect waste products in specifically assigned waste containers.
<ol>
	<li>Dissolve an appropriate amount of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) powder in PBS to obtain a 10x stock solution at 5 mg/mL. Sterilize it by filtration with a 0.22 &#181;m filter and store aliquots at -20 &#176;C.</li>
	<li>Plate cells on 24-well plates (3 wells per experimental condition per plate). Do not forget to reserve 3 empty wells per plate for the spectrophotometer blanks.</li>
	<li>Incubate cells at 37 &#176;C for 22-24 h prior to uranium exposure to let cells attach.</li>
	<li>Prepare uranyl-containing media as described in section 3.</li>
	<li>Replace growth medium in the cell-seeded plates with uranyl-containing media. Incubate for 24 h.</li>
	<li>Calculate the necessary volume of 1x MTT solution as follows: 
	(Number of experimental conditions + blank) x 3 (triplicates) x 0.4 mL/well + 10% volume for pipetting losses.</li>
	<li>Prepare the required volume of 1x MTT solution by diluting the 10x stock MTT solution in Eagle&#39;s Minimum Essential Medium (EMEM) without phenol red. 
	NOTE:For a successful colorimetric assay, MTT should be protected from light. It is recommended to dilute MTT into EMEM rather than DMEM in order to limit the background signal.</li>
	<li>Remove uranyl-containing media from the wells, wash them with PBS and add 400 &#181;L of MTT solution to each well (including 3 empty wells for blanks). Incubate 2.5 h at 37 &#176;C in the dark. Verify under microscope that dark purple formazan crystals have formed inside viable cells.</li>
	<li>Discard the MTT solution and add 220 &#181;L of dimethyl sulfoxide (DMSO) per well. Wrap plates in aluminum foil to protect them from the light and gently agitate for 10 min to dissolve formazan crystals.</li>
	<li>Use a microplate spectrometer reader to determine the optical density (OD) at 570 nm (formazan specific) and 690 nm (background). For each well, subtract 690 nm OD from 570 nm OD to adjust for possible non-specific OD value fluctuation due to scratches or turbidity22.</li>
	<li>Calculate the blank-corrected OD for each well by subtracting mean OD of the blank from the OD obtained in the previous step. Determine the mean OD value for each experimental condition.</li>
	<li>Set mean OD of the control condition ([UO22+] = 0 mM) to 100% viability and calculate the corresponding percentage of cell viability for other tested uranyl concentrations. Plot a dose-response curve. Evaluate the Cytotoxicity Index 50 (CI50), defined as the uranyl concentration leading to 50% cell death after 24h exposure.</li>
</ol>
5. Analysis of Osteoclast Differentiation in the Presence of U(VI)
<ol>
	<li>Differentiation of RAW 264.7 cells in the presence of uranyl and TRAP (Tartrate -Resistant Acid Phosphatase) staining.
	<ol>
		<li>Plate RAW 264.7 cells on a 24-well plate, 3 wells per experimental condition. Incubate at 37 &#176;C for approximately 6 h, the time to prepare and equilibrate the uranyl-containing exposure media.</li>
		<li>Prepare differentiation media with desired uranyl concentrations as described in section 3 (do not forget to add GST-RANKL to media).</li>
		<li>Gently replace basic medium in wells with uranyl-containing medium. This day is considered as day 0 of osteoclastic differentiation.</li>
		<li>Change the medium on day 2 or 3 of osteoclastic differentiation by repeating steps 5.1.2 and 5.1.3. Multinucleated osteoclasts should appear between day 3 and 4.</li>
		<li>Perform the TRAP staining with the commercially available Leukocyte acid phosphatase kit following the manufacturer&#39;s instructions. 
		NOTE:Tartrate-resistant acid phosphatase (TRAP) also called acid phosphatase 5, tartrate resistant (ACP5) is highly expressed in mature osteoclasts and extensively used as a marker for osteoclast differentiation. Therefore, TRAP staining is performed to visualize osteoclasts. Depending on experimental conditions and the rate of osteoclastogenesis, it could be done on day 4 or day 5 of the differentiation process.</li>
		<li>Keep TRAP-stained wells in PBS at 4 &#176;C for further analysis (they can be maintained in these conditions up to 3 or 4 weeks).</li>
	</ol>
	</li>
	<li>Osteoclast size/morphology analysis.
	<ol>
		<li>Acquire an image of the whole surface of each well. The resolution of the image should be sufficient to distinguish cell nuclei. Consider TRAP-positive cells with 3 or more nuclei, as osteoclasts.</li>
		<li>Open an image of one well in the ImageJ software: &#34;File&#34; &#8594; &#34;Open&#34;.</li>
		<li>Verify the scale units in the upper left corner of the image. For the relative quantification, any unit can be used. If the absolute value of osteoclast size is necessary, convert scale units to &#181;m: &#34;Image&#34; &#8594; &#34;Properties&#34;. Check the box &#34;global&#34; in the pop-up window in order to have new scale units applied to all images opened later. 
		NOTE: If a microscope was used for image acquisition, the pixel size in &#181;m is indicated for each objective in the imaging software.</li>
		<li>Specify measurement parameters to be analyzed: &#34;Analyze&#34; &#8594; &#34;Set Measurements&#34;. In the Set Measurements window, check &#34;Area&#34; and &#34;Display Label&#34; boxes and uncheck all the others boxes.</li>
		<li>Open the ROI (region of interest) manager: &#34;Analyze&#34; &#8594; &#34;Tools&#34; &#8594; &#34;ROI Manager&#34;.</li>
		<li>In the main window, pick the rectangular selection tool. Use it anywhere in the image to outline a sector of about 1,000 x 1,000 &#181;m. This selection now defines the first region in which osteoclast size will be analyzed.</li>
		<li>In the ROI Manager window, click on &#34;Add&#34; button to add this region of interest to the ROI manager and save it by clicking on &#34;More&#34; &#8594; &#34;Save&#34;. The saved ROI can now be reloaded to the ROI manager anytime by clicking on &#34;More&#34; &#8594; &#34;Open&#34;.</li>
		<li>Repeat steps 5.2.5 - 5.2.6 to define 4 additional regions for osteoclast size analysis (Figure 2A and 2D). 
		NOTE: The defined regions will be used to analyze osteoclast sizes in all the images.</li>
		<li>Create a copy of one of the 5 regions to be analyzed: chose this ROI in the main the ROI manager (the corresponding selection will appear on the main image) &#8594; right click inside the selection &#8594; &#34;Duplicate&#34;. Use this duplicate for further analysis.</li>
		<li>In the ROI Manager window, check boxes &#34;Show Al&#34;l and &#34;Labels&#34;.</li>
		<li>In the main window, choose the polygon selection tool to manually outline one of the osteoclasts in the selection. Add this outline to the ROI manager by clicking on &#34;Add&#34; in the ROI manager window. Proceed similarly for all osteoclasts that lie completely inside the selected region (Figure 2B and 2E).</li>
		<li>In the ROI Manager window, select all the outlines and measure their surface by clicking on &#34;Measure&#34;. Transfer the measurements that have appeared in a new window (Results window, Figure 2C and 2F) to a spreadsheet. 
		NOTE: At this point, the region of analysis with all the osteoclasts outlines could be saved as an independent image: &#34;File&#34; &#8594; &#34;Save As&#34; format of choice.</li>
		<li>Repeat steps 5.2.8 - 5.2.12 for the remaining regions of analysis of the current image. Then, repeat the whole procedure for all the other images.</li>
	</ol>
	</li>
	<li>Osteoclast number analysis with ImageJ software. 
	NOTE: As osteoclast distribution in the well is rarely homogenous, perform the counting on the whole well rather than on few selected zones. Given that osteoclasts are heterogeneous in form and size, no automated counting cell method is practicable.
	<ol>
		<li>Open a whole-well image in the ImageJ software (for this analysis, use images from previous sub-section).</li>
		<li>Open the cell counter window: &#34;Plugins&#34; &#8594; &#34;Analyze&#34; &#8594; &#34;Cell Counter&#34;. Click on &#34;Initialize&#34; and check &#34;Type 1&#34; box (or whatever type number you prefer). Verify that &#34;Show Number&#34; box is checked and &#34;Delete Mode&#34; box is unchecked.</li>
		<li>On the image, click on one osteoclast: colored dot and a number will appear where clicked. Proceed similarly for all osteoclasts. Save the markers regularly by clicking on &#34;Save Markers&#34; in the cell counter window 
		NOTE: If the last point has to be removed from the count, click on &#34;Delete&#34; in the cell counter window. If there is more than one point to remove, check the &#34;Delete Mode&#34; box and indicate all invalid points by clicking on them. Then uncheck the &#34;Delete Mode&#34; box to continue the count.</li>
		<li>Display the result of counting by clicking on Results in the cell counter window. Transfer the final counting results to a spreadsheet.</li>
	</ol>
	</li>
</ol>
6. Analysis of Osteoclast Function in the Presence of U(VI)
<ol>
	<li>Pit resorption assay.
	<ol>
		<li>Plate RAW 264.7 cells on a 24-well osteo assay plate, which provides a synthetic inorganic osteomimetic surface that could be resorbed by osteoclasts. 
		NOTE:In order to let cells attach to the biomimetic surface, it is often better to plate them the day before adding the differentiation medium.</li>
		<li>Differentiate RAW 246.7 cells into osteoclasts as described in section 5 (steps 5.1.2 - 5.1.4)</li>
		<li>On day 5 of osteoclastogenesis, remove osteoclasts from the bone mimetic surface by replacing uranyl-containing medium with 10% bleach solution. Incubate for 5 min at room temperature. Wash wells twice with deionized water and let them air-dry.</li>
		<li>Add a 1% Alizarin Red S sodium salt solution to the wells to stain calcium salts in the non-resorbed areas. Remove Alizarin solution after 10-15 s of incubation and gently wash the wells 3-4 times with water. Let the wells air-dry. 
		NOTE: The staining procedure described above is designed to enhance contrast between the resorbed and the non-resorbed areas in order to facilitate consecutive analysis. This procedure must be done carefully because if wells are not completely dry before staining, parts of the remaining bone-mimetic surface could be accidentally removed. Moreover, if Alizarin solution is left too long in the wells, a persistent non-specific staining will appear. It should be noted that the test described in this section relates to the effect of uranium on both osteoclastic differentiation and function. To study the effect of uranium on resorption independently of osteoclast formation, RAW 264.7 cells can be treated with uranyl-containing medium for 24 hours, only once mature osteoclasts are formed (at day 4 or 5 of the differentiation process) 23.</li>
	</ol>
	</li>
	<li>Pit resorption analysis.
	<ol>
		<li>Acquire an image of the whole surface of each well of the osteo assay plate. 
		NOTE: Image acquisition methods can vary but should be consistent throughout replicate experiments. They can include a picture taken with a macro objective of a fixed camera, a high-resolution scan or a mosaic image made with an automated high throughput microscope.</li>
		<li>Begin analysis by repeating steps 5.2.2 to 5.2.4 from the previous section.</li>
		<li>Convert an RGB image to a greyscale 8-bit format: &#34;Image&#34; &#8594; &#34;Type&#34; &#8594; &#34;8 bit&#34;.</li>
		<li>In the main window, select the oval selection tool. Use it to draw a circle outlining all the surface of the well to be analyzed for resorption.</li>
		<li>Save this new ROI as in step 5.2.6.</li>
		<li>To facilitate threshold selection, clear all the features outside the defined ROI: &#34;Edit&#34; &#8594; &#34;Clear Outside&#34;. 
		NOTE: At this point, it is often better to make a few copies of the image: deselect ROI by clicking outside the selected zone&#160;&#8594; right click on the image &#8594; &#34;Duplicate&#34;. This action will help avoid a repetition of the image treatment described above if the threshold chosen in the next step is unsatisfactory.</li>
		<li>Select &#34;Image&#34; &#8594; &#34;Adjust&#34; &#8594; &#34;Threshold&#34;. In the Threshold window, use the slider bar to choose a suitable threshold. All resorbed areas should be below the threshold (white) and non-resorbed, above (red). To finalize the threshold selection, click on &#34;Apply&#34; in the Threshold window.</li>
		<li>Save the final binary image: &#34;File&#34; &#8594; &#34;Save As&#34; &#8594; format of your choice.</li>
		<li>Define measurements to be taken in the region of interest: &#34;Analyze&#34; &#8594; &#34;Set Measurements&#34;. In the Set Measurements window, check &#34;Area&#34;, &#34;Area fraction&#34; and &#34;Limit to threshold&#34; boxes and uncheck all the others.</li>
		<li>In order to obtain the fraction of &#34;resorbed&#34; area directly in the Results window, invert the binary image (&#34;Edit&#34; &#8594; &#34;Invert&#34;). Ensure that the main ROI is selected on the final binary image (otherwise resorbed area fraction will be calculated based on the surface of the whole image window). Measure resorbed area fraction by either clicking on &#34;Measure&#34; in the ROI manager window or on &#34;Analyze&#34; &#8594; &#34;Measure&#34;. Save the result.</li>
	</ol>
	</li>
</ol>

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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>

The authors have nothing to disclose

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>&amp;alpha;-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>&amp;nbsp;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 cm&lt;sup&gt;2&lt;/sup&gt;</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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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 ...

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Universidad de Cartagena UDEC

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8.1K Views.  Université Nice Sophia-Antipolis. 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.

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

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted through ion exchange. The resultant production bleed water (PBW) contains contaminants such as arsenic and other heavy metals. Samples of PBW from an active ISR uranium facility were treated with cupric oxide nanoparticles (CuO-NPs). CuO-NP treatment of PBW reduced priority contaminants, including arsenic, selenium, uranium, and vanadium. Untreated and CuO-NP treated PBW was used as the liquid component of the cell growth media and changes in viability were determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human embryonic kidney (HEK 293) and human hepatocellular carcinoma (Hep G2) cells. CuO-NP treatment was associated with improved HEK and HEP cell viability. Limitations of this method include dilution of the PBW by growth media components and during osmolality adjustment as well as necessary pH adjustment. This method is limited in its wider context due to dilution effects and changes in the pH of the PBW which is traditionally slightly acidic&#160;however; this method could have a broader use assessing CuO-NP treatment in more neutral waters.

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

Production bleed water (PBW) was treated with cupric oxide nanoparticles (CuO-NPs) and cellular toxicity was assessed in cultured human cells. The goal of this protocol was to integrate the native environmental sample into a cell culture format assessing the changes in toxicity due to CuO-NP treatment.

In situ recovery (ISR) is the predominant method of uranium extraction in the United States. During ISR, uranium is leached from an ore body and extracted ...

Environment

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

Cancer Research

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Medicine

Effect of Anti-c-fms Antibody on Osteoclast Formation and Proliferation of Osteoclast Precursor In Vitro

Bone remodeling is a complex process and it involves periods of deposition and resorption. Bone resorption is a process by which bone is broken down by osteoclasts in response to different stimuli. Osteoclast precursors differentiate into multinuclear osteoclasts in response to macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor Kappa-B ligand (RANKL). Under pathologic conditions, the cytokine profile is different and involves a mixture of inflammatory cytokines. Tumor necrosis factor alpha (TNF-&#945;) is one of the most important cytokines as it is found in large amounts in areas involved with inflammatory osteolysis. The purpose of this protocol is to provide a method by which murine bone marrow is isolated to generate osteoclasts through induction with M-CSF and either RANKL or TNF-&#945; which will be subsequently inhibited by increasing doses of anti-c-fms antibody, the receptor for M-CSF. This experiment highlights the therapeutic value of anti-c-fms antibody in diseases of inflammatory bone resorption.

This protocol includes dissection of murine long bones and bone marrow isolation, to generate bone marrow macrophages and produce osteoclasts using macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor Kappa-B ligand (RANKL) or tumor necrosis factor alpha (TNF-&#945;) and their arrest by anti-c-fms antibody, M-CSF receptor.

Bone remodeling is a complex process and it involves periods of deposition and resorption. Bone resorption is a process by which bone is broken down by ...

Immunology and Infection

Osteoclast Derivation from Mouse Bone Marrow

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

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

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

Biology

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.

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.

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

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

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

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

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

Resorption Pit Assay: An In Vitro Technique to Quantify Calcium Resorption Activity of Mature Osteoclasts using Calcium Phosphate-Coated Culture Plates

Source: Cen, W., et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/64016">A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro.</a> J. Vis. Exp. (2022)
In this video, we describe an in vitro osteoclastic resorption assay on calcium phosphate-coated cell culture plates cultured with human peripheral blood mononuclear cell-derived osteoclasts. The osteoclasts form a characteristic actin ring on the calcium phosphate-coated matrix of the cell culture plate and degrade the matrix within the actin ring region to release calcium and phosphate ions, resulting in the formation of distinct resorption pits within the degraded matrix.

Source: Cen, W., et al. A Simple Pit Assay Protocol to Visualize and Quantify Osteoclastic Resorption In Vitro. J. Vis. Exp. (2022)
In this video, we ...

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Assay Techniques

Cell-based assays

In Vitro Mineralization Assay: A Colorimetric Method to Quantify Osteoblast Calcium Deposits on Bone Graft Substitute by Alizarin Red S Staining and Extraction

Source: Kampleitner, C. et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/58077">Biological Compatibility Profile on Biomaterials for Bone Regeneration.</a> J. Vis. Exp. (2018)
In this video, we describe a method for the detection of osteoblast-secreted calcium deposits using an anionic dye, Alizarin Red S. The cultured osteoblasts are first stained and then the dye is recovered for colorimetric quantification of calcium deposits or the degree of osteoblast mineralization.

Source: Kampleitner, C. et al. Biological Compatibility Profile on Biomaterials for Bone Regeneration. J. Vis. Exp. (2018)
In this video, we describe a ...

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

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Chapters in this video

Removal of Trace Elements by Cupric Oxide Nanoparticles from Uranium In Situ Recovery Bleed Water and Its Effect on Cell Viability

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Effect of Anti-c-fms Antibody on Osteoclast Formation and Proliferation of Osteoclast Precursor In Vitro

Osteoclast Derivation from Mouse Bone Marrow

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

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

Resorption Pit Assay: An In Vitro Technique to Quantify Calcium Resorption Activity of Mature Osteoclasts using Calcium Phosphate-Coated Culture Plates

In Vitro Mineralization Assay: A Colorimetric Method to Quantify Osteoblast Calcium Deposits on Bone Graft Substitute by Alizarin Red S Staining and Extraction