Osteoclast Activity Assay Substrate

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Osteoclast Activity Assay Substrate For Research Use Only OSCOTECT INC. #3201 Trade Tower Samsung-dong 159, Kangnam-ku Seoul 135-729, Korea Tel: +82-2-6000-7666 / Fax: +82-2-6000-7667 [email protected] http://www.oscotec.com -1- Osteoclast Activity Assay Substrate (OAASTM) You don’t need to make dentin / bone slice laboriously and use SEM and more for osteoclast study! As a culture dish with its bottom coated with calcium-phosphate thin film that would become substrate for osteoclasts, OAAS is designed to measure bone resorption activity of the osteoclasts. Bone resorption activity of the osteoclasts can be measured by direct observation under phase contrast microscopy without resort to a scanning electron microscope (SEM). Since the substrate is transparent, cells can be directly observed even during culture, and the area of resorption can be easily measured by using an image analyzer. -2- - Figure 1, 2: Resorption areas in a culture dish after removing osteoclasts - Figure 3: TRAP staining of osteoclasts in situ culture dish and resorption area - Figure 4: SEM picture of an osteoclast and resorption area How to Use OAASTM 1. Open an OAAS package in clean area such as clean bench. 2. Place the appropriate amount of culture media containing cells into each well of the OAAS plate. 3. Culture cells according to the experimental protocol. 4. Attached cells and resorption area can be observed with a phase contrast microscope, even during the process of culture. 5. Remove the culture media from the OAAS plate on completion of culture. 6. Wash the OAAS plate with saline and leave it in a solution of 5% sodium hypochlorite for 5 minutes in order to detach the cells. 7. Wash the OAAS plate again with saline, and then dry it. Observe the resorption area. -3- Image Analysis for OAASTM You can measure the degree of resorption by osteoclasts in OAAS using one of the following methods. Method 1: Get images with a CCD camera attached to a microscope and a capture or grabber card in your computer. Then, measure the number and area of resorption in the images. The image analysis softwares used frequently are Image-Pro Plus, Scion Image (for MacOS and Windows), and Adobe Photoshop. Method 2: Get images with a digital camera attached to a microscope (low magnitude, 1.25 × 10). Then, measure the number and area of resorption in the images. Method 3: Another way to acquire images is to scan microscopic pictures with a desktop scanner. Preferred Method of Image Analysis 1. Divide a well to the proper size for capture. 2. Capture divided areas of the well serially. Forty-fold magnification would be appropriate for measurement (Figure 5). 3. Open an image file in Jasc Paint Shop Pro™. 4. Capture resorption areas with the Magic Wand (under the " Tool" Options of Magic Wand, which is activated with a click of the right mouse button. We suggest that you set Tolerance = 11 and Feather = 2 or 3). When all the resorption areas are captured in an image, copy the areas with Ctrl-C and paste them in a new image file with Ctrl-V. -4- 5. Adjust the brightness and contrast using Ctrl-B, making the resorption areas dark, and save the image file under a new name (Figure 6). 6. Open the image file in Image-Pro Plus™ (Media Cybernetics, Version 3.0). 7. Select the Count/Size… command from the Measure menu. Click the Automatic Dark Objects radio button in the Intensity Range Selection group box, and then click Count. (Figure 7.) 8. From the Measure menu, select the Single Variable Class command. The Single Variable Class dialog box appears. If you want to see only the value of the resorption area, leave the Number of Bins field at 2. It you want to see the range of values, set the Number of Bins field to the number of ranges that you want to divide. 9. Click OK. The Classification data window appears. This window lists the number of objects in each class, the percentage of objects in each class, the range values, and sum of the values. You can select the items that you want to get from the View menu. 10. From the File menu, select DDE to Excel command in order to send the data to Microsoft Excel. Figure 5 Figure 6 - Figure 5: An image of a divided area of OAAS after several days culture captured by digital camera. (Clear areas are resorbed.) - Figure 6: Image of Figure 5 processed by imaging application. (Black areas are resorbed.) -5- Figure 7 - Figure 7: Calculating the area of resorption using Image pro plus™. Papers where OAASTM was used ◆ gp130-Mediated Signaling Is Necessary for Normal Osteoblastic Function in Vivo and in Vitro Endocrinology 145: 1376–1385, 2004 ◆ Transcriptional Activity of Nuclei in Multinucleated Osteoclasts and Its Modulation by Calcitonin Endocrinology 143: 1913–1921, 2002 ◆ Attenuation of bone mass and increase of osteoclast formation in decoy receptor 3transgenic mice J. Biol. Chem. 282: 2346-2354, 2007 ◆ Regulation of osteoclast differentiation by the redox-dependent modulation of nuclear import of transcription factors Cell Death. Differ. 13: 1138-1146, 2006 ◆ Osteoclastogenesis in the Nonadherent Cell Population of Human Bone Marrow Is I hibit d b hBMP 2 Al T th ith hVEGF I J O th R 24 29 36 -6- 2006 ◆ Conjugated linoleic acid inhibits osteoclast differentiation of RAW264.7 cells by modulating RANKL signaling J Lipid Res 47: 1739–1748 2006 ◆ Bimodal actions of reactive oxygen species in the differentiation and bone-resorbing functions of osteoclasts FEBS Letters 580: 5661–5665, 2006 ◆ Effects of mushroom, Pleurotus eryngii, extracts on bone metabolism Clinical Nutrition 25: 166–170, 2006 ◆ Alternative bisphosphonate targets and mechanisms of action Biochem. Bophys. Res. Commun.328: 746–750, 2005 ◆ Inhibition of RANKL-Induced Osteoclastogenesis by (−)-DHMEQ, a Novel NF-_B Inhibitor, Through Downregulation of NFATc1 J. Bone Miner. Res, 20: 653–662, 2005 ◆ Ascorbic acid inhibits osteoclastogenesis of RAW264.7 cells induced by receptor activated nuclear factor kappaB ligand (RANKL) in vitro. J. Endocrinol. Invest. 28: 253-260, 2005 ◆ Ascorbic acid inhibits the formation and function of osteoclasts from RAW264.7 cells induced by receptor activated nuclear factor kappaB ligand in vitro Zhonghua Yi Xue Za Zhi. 84: 102-2106, 2004 ◆ Decoy receptor 3 (DcR3) induces osteoclast formation from monocyte/macrophage lineage precursor cells Cell Death and Differentiation 11: S97–S107 2004 ◆ Potent antiarthritic properties of a glucocorticoid derivative, NCX-1015, in an experimental model of arthritis PNAS 99: 1677–1682, 2002 ◆ Cyclic Adenosine Monophosphate/Protein Hormone/Parathyroid Hormone–Related Kinase Protein A Mediates Receptor Parathyroid Regulation of Osteoclastogenesis and Expression of RANKL and Osteoprotegerin mRNAs by Marrow Stromal Cells Bone Miner. Res. 17: 1667–1679, 2002 ◆ Effect of Rehmannia glutinosa Libosch extracts on bone metabolism. Clinica Chimica Acta 334: 185-195, 2003 -7- ◆ Effects of Two Herbal Extracts on the Bone Metabolism. J Bone Miner Res 16: S413, SU436 (Abstract), 2001 ◆ High Extracellular Calcium Alone Stimulates Osteoclast Formation but Inhibits in the Presence of 1,25-(OH)2VitD3. J Bone Miner Res 16: S503, M293 (Abstract), 2001 ◆ Osteoclastogenesis is enhanced by activated B cells but suppressed by activated CD8(+) T cells. ◆ Eur J Immunol 31: 2179-2188, 2001 Regulation of Osteoclast Differentiation by Activated Lymphocytes. J Bone Miner Res 15: S511, M229 (Abstract), 2000 ◆ Effects of Nitric Oxide on the Osteoclast Differentiation and Activity in Mouse Bone Marrow Cells in Culture. J Bone Miner Res 15:S512, M234 (Abstract), 2000 ◆ Formation of Mature Osteoclasts from Human Peripheral Blood Mononuclear Cells (PBMC) in the Absence of Stromal / Osteoblastic Cells by RANK Ligand. J Bone Miner Res 15:S512, M237 (Abstract), 2000 ◆ Prostaglandin E2 Stimulates Osteoclast Formation through Direct Effects on Osteclast Precursors. J Bone Miner Res 15:S513, M239 (Abstract), 2000 ◆ Human Osteoclast Differentiation is Regulated by Cell or Serum Factors in Addition to CSF-1 and RANKL. J Bone Miner Res 15:S513, M241 (Abstract), 2000 FAQ >> How to remove osteoclasts in order to measure the resorption area? In order to remove osteoclast after its cultivation from OAAS, eliminate the culture fluid, treat culture in 5% NaOCl for 5minutes, wash with distilled water, and observe after drying. -8- >> What is the method for measuring the resorption area, and does OCT handle related measuring equipments? You can use the following methods to measure the resorption pit of osteoclasts. However, we do not handle related measuring equipments. Method 1: You can observe the resorption pit of OAAS directly if the optical microscope is equipped with a CCD camera connected to a PC with a capture card or grabber card. Capture the image directly from the microscope using the PC, then use the following image analyzing program to measure the number or area of the resorption pit. Most widely used softwares are Image-Pro Plus, Scion Image (Windows, MacOS), and PhotoShop. Method 2: If your microscope has a low-magnifying lens (1.25 × 10), you can observe the OAAS resorption pit and estimate the number and area using the image analyzing program on the data saved on the digital camera. Method 3: Use a scanner to scan the picture from the microscope. Then continue with analysis by using the same software mentioned in method 1. >> Do osteoclasts disappear when TRAP staining? After resorption is complete, osteoclast cells may be separated from the OAAS plate by apoptosis (cell death) if left alone too long. During a normal active state, however, the TRAP dye does not make the osteoclast disappear. No problems should arise during fixation as long as one follows the TRAP dye manufacturing company’s instructions. >> What is the required culture period for mature cell and precursor cell using OAAS? It takes about 24 hours for both mature and precursor cells. It depends on the cell differentiation stage, which is 7 to 10 days after splitting culture. More time is required for cells to attach on OAAS plate compared to normal plates, and the time varies depending on -9- laboratory conditions. Osteoclasts can be observed during OAAS plate culture, allowing one to validate and make time adjustments through the microscope during culture. >> How to estimate Osteoclast resorption activity through 3D? Currently, 3D observation is unavailable.