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>&#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 ...

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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

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). We use an in vitro myelination assay, an established model for studying CNS myelination in vitro. To do this, oligodendrocyte precursor cells (OPCs) are added to the purified primary rodent dorsal root ganglion (DRG) neurons to form myelinating co-cultures. In order to specifically interrogate the roles that particular proteins expressed by oligodendrocytes exert upon myelination we have developed protocols that selectively transduce OPCs using the lentivirus overexpressing wild type, constitutively active or dominant negative proteins before being seeded onto the DRG neurons. This allows us to specifically interrogate the roles of these oligodendroglial proteins in regulating myelination. The protocols can also be applied in the study of other cell types, thus providing an approach that allows selective manipulation of proteins expressed by a desired cell type, such as oligodendrocytes for the targeted study of signaling and compensation mechanisms. In conclusion, combining the in vitro myelination assay with lentiviral infected OPCs provides a strategic tool for the analysis of molecular mechanisms involved in myelination.

Production and Use of Lentivirus to Selectively Transduce Primary Oligodendrocyte Precursor Cells for In Vitro Myelination Assays

Here we present protocols that offer a flexible and strategic foundation for virally manipulating oligodendrocyte precursor cells to overexpress proteins of interest in order to specifically interrogate their role in oligodendrocytes via the in vitro model of central nervous system myelination.

Myelination is a complex process that involves both neurons and the myelin forming glial cells, oligodendrocytes in the central nervous system (CNS) and ...

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 development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

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

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a population of rapidly cycling intestinal stem cells (ISCs) characterized by their expression of the Lgr5 gene. These are supported by a quiescent stem cell population, marked by Bmi-1 expression, capable of replacing them in the event of injury. Investigating the interactions between these populations is crucial to understanding their roles in disease and cancer. The ISCs exist within crypts on the intestinal surface, these niches support the ISC in replenishing the epithelia. The interaction between active and quiescent ISCs likely involves other differentiated cells within the niche, as it has previously been demonstrated that the &#8216;&#8216;stemness&#8217;&#8217; of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. Using conditional cre-lox mouse models we tested the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. Here we describe the techniques and analysis undertaken to characterize the intestine and demonstrate that the Paneth cells play a crucial role within the ISC niche in aiding recovery following substantial insult.

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

Intestinal epithelial stem cells (ISCs) are intermingled with Paneth cells. These cells are differentiated progeny of the ISC, which support the ISCs and provide antibacterial protection. Here we demonstrate how we used transgenic conditional mouse models to establish that Paneth cells play a crucial role in maintaining the intestinal epithelia.

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

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

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

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live Drosophila Ovarian Tissue

We provide a protocol for imaging intracellular pH of an epithelial stem cell lineage in live Drosophila ovarian tissue. We describe methods to generate transgenic flies expressing a pH biosensor, mCherry::pHluorin, image the biosensor using quantitative fluorescence imaging, generate standard curves, and convert fluorescence intensity values to pH values.

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

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 and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

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

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

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

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

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 formation of new blood vessels and involve several signal transduction pathways. Here we show that the tube formation assay is a simple in vitro method to evaluate the impact of natural products on angiogenesis and to investigate the molecular mechanisms involved. In particular, in the presence of the water extract of Ruta graveolens (RGWE), endothelial cells are no longer able to form a cell-cell network and that the RGWE effects on human umbilical vein endothelial cell (HUVEC) tube formation is abolished by the constitutive activation of MEK.

Here, we evaluate the effects of the water extract of Ruta graveolens on vessel network formation by using a tube formation assay on a gelled basement matrix.

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

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor chemicals (EDCs). These chemicals may alter the normal balance of endocrine systems and lead to adverse effects, as well as an increasing number of hormonal disorders in the human population or disturbed growth and reduced reproduction in the wildlife species. For some EDCs, there are documented health effects and restrictions on their use. However, for most of them, there is still no scientific evidence in this sense. In order to verify potential endocrine effects of a chemical in the full organism, we need to test it in appropriate model systems, as well as in the fruit fly, Drosophila melanogaster. Here we report detailed in vivo protocols to study endocrine disruption in Drosophila, addressing EDC effects on the fecundity/fertility, developmental timing, and lifespan of the fly. In the last few years, we used these Drosophila life traits to investigate the effects of exposure to 17-&#945;-ethinylestradiol (EE2), bisphenol A (BPA), and bisphenol AF (BPA F). Altogether, these assays covered all Drosophila life stages and made it possible to evaluate endocrine disruption in all hormone-mediated processes. Fecundity/fertility and developmental timing assays were useful to measure the EDC impact on the fly reproductive performance and on developmental stages, respectively. Finally, the lifespan assay involved chronic EDC exposures to adults and measured their survivorship. However, these life traits can also be influenced by several experimental factors that had to be carefully controlled. So, in this work, we suggest a series of procedures we have optimized for the right outcome of these assays. These methods allow scientists to establish endocrine disruption for any EDC or for a mixture of different EDCs in Drosophila, although to identify the endocrine mechanism responsible for the effect, further essays could be needed.

Methods to Test Endocrine Disruption in Drosophila melanogaster

Endocrine disruptor chemicals (EDCs) represent a serious problem for organisms and for natural environments. Drosophila melanogaster represents an ideal model to study EDC effects in vivo. Here, we present methods to investigate endocrine disruption in Drosophila, addressing EDC effects on fecundity, fertility, developmental timing, and lifespan of the fly.

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 stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

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