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

The overall goal of this procedure is to investigate the effects of natural uranium exposure on osteoclast viability, differentiation and function. This method can help answer key questions in the environmental toxicity field about the impact of uranium exposure on the bone physiology and metabolism. The main advantage of this technique is that it provides an interesting new tool for the screening of uranium originators.

When the RAW 264.7 cell culture has reached 85 to 90%confluency, use a cell scraper to mechanically lift the cells from the bottom of the cell culture flask and transfer the cells into a sterile 50-milliliter conical tube. Pipette the cell solution a few times to break up the clumps and count the resulting single-cell suspension. Dilute the cells to a one times 10-to-the-fourth cells-per-milliliter concentration in alpha-modified Minimum Essential Medium, supplemented with L-glutamine and 5%fetal bovine serum and add one milliliter of cells per well to a 24-well cell culture plate for a six-hour incubation at 37 degrees Celsius.

An important parameter for the reproducibility of the osteoclast differentiation and reabsorption assay is the cell seeding density. Pay particular attention to the cell counting, cellular suspension preparation and its homogeneity before plating the cells. To prepare the urinal salt solution, starting from a sterile cell culture-tested 7.5%sodium bicarbonate aqueous solution, first prepare and cool 100 millimolar bicarbonate in water on ice and add one volume of 100 millimolar urinal acetate solution, drop by drop, into nine volumes of the ice-cold bicarbonate solution with gentle shaking.

Allow the solution to equilibrate at room temperature. After three hours, dilute the appropriate volume of urinal solution, drop by drop, into differentiation medium containing 50 nanograms per milliliter of RANKL until the desired urinal working concentration is achieved and allow another three hours of equilibration at room temperature. The long incubation steps after each dilution of the urinal salt are essential for stabilization of the urinal complexes in the solutions.

At the end of the second equilibration period, gently replace the medium in each well with one milliliter of urinal treated or control medium as appropriate and return the cells to the incubator. Multinucleated osteoclasts should appear between days three and four of culture. To assess the size and morphology of the newly differentiated osteoclasts, stain the cells with tartrate-resistant acid phosphatase, according to the manufacturer's instructions and image the whole surface of each well at a resolution sufficient for distinguishing cell nuclei.

When all of the wells have been imaged, open an image of one well with open-source ImageJ software and verify the scale units. Then check the Global box in the popup window to apply the new scale units to all of the later images. Under the Analyze menu, select Set Measurements and check the Area and Display label boxes.

Uncheck all of the other boxes. Under the Analyze menu, open Tools then the Region of Interest Manager and select the rectangular selection tool in the main ImageJ window. Outline a region of interest of about 1, 000-by-1, 000 micrometers and click the Add button to add the selection to the Region of Interest Manager.

Then click More, Save to save the region. To create a copy of one of the five regions to be analyzed, select the region of interest in the Region of Interest Manager and right-click inside the selected area to duplicate the region. Check the Show All and Labels boxes and select the polygon selection tool to manually outline one of the osteoclasts in the duplicated region of interest.

To add this outline to the Region of Interest Manager, click Add in the Region of Interest Manager window. When all of the osteoclasts have been added, select all of the outlines and click Measure to measure their surfaces. Then transfer the measurements that appear in the results window to a spreadsheet.

To assess the function of urinal treated osteoclasts, one day before adding the differentiation medium, plate RAW cells from an 85 to 90%confluent cell culture on to a 24-well osteo assay plate in growth medium, as just demonstrated. The next day, gently replace the supernatant with differentiation urinal containing medium and allow the RAW cells to differentiate into osteoclasts, as just demonstrated. On day five of osteoclastogenesis, replace the urinal-containing medium with 10%bleach solution to remove the cells from the bone mimetic surface.

After five minutes at room temperature, wash the wells two times with deionized water and let them air dry. Next, stain the calcium salts in the non-resorbed areas of each well with a 1%Alizarin Red S sodium salt solution. After 10 to 15 seconds, remove the Alizarin solution and gently wash the wells three to four times with water.

To convert an RGB image of the Alizarin-stained wells to a grayscale, 8-bit format, under the Image menu, select Type and 8-bit. Then, using the oval selection tool, outline the entire surface of the well to be analyzed for resorption and save the region of interest, as just demonstrated. To facilitate threshold selection, select Edit and Clear Outside to clear all of the features outside of the defined region of interest.

Duplicate the region of interest as just demonstrated and select Adjust Threshold under the Image menu, adjusting the slide bar to select an appropriate threshold. All of the resorbed areas should be above the threshold and appear white, while all of the non-resorbed areas should be below the threshold and appear red. To finalize the threshold selection, click Apply in the Threshold window and save the final binary image in the appropriate format of interest.

To define the measurements to be taken within the region of interest, under the Analyze menu, select Set Measurements and check Area, Area Fraction and Limit to Threshold boxes and uncheck all of the other boxes. Then to obtain the fraction of resorbed area directly within the results window, invert the duplicated binary image by selecting Invert under the Edit menu and taking care that the main region of interest is selected on the final binary image and measure the resorbed area fraction. The changes in number and size of newly differentiated osteoclasts in response to uranium are easily visible in composite images of whole wells, as well as in enlarged pictures of select regions of interest.

The size of the osteoclasts grown under different culture conditions can be analyzed as just demonstrated by outlining all of the osteoclasts within each region and measuring the area of each outlined cell. To investigate the impact of uranium on osteoclast resorption activity, RAW 264.7 cells are plated and differentiated on osteo mimetic surface plates. At the end of the assay, the osteoclasts are removed and the non-resorbed areas are stained and imaged as demonstrated.

The images are then converted to 8-bit grayscale and thresholded to facilitate calculation of the resorbed areas. This procedure can also be applied to the study of the impact of other heavy metals on osteoclastogenesis.