Our protocol describes a simple and reliable method for an osteoclastic resorption assay. The use of calcium phosphate coated cell culture plates can be easily prepared and visualized using lab available materials. In contrast to opaque bone or dentin slices, the calcium phosphate coating allows visualization of resorption pits and cells at the same time.
In this protocol, we use PBMCs for osteoclast differentiation. However, it can also be applied to other cell types like bone marrow derived monocytes or Raw 264.7 cells. First take a flat bottom 96 well cell culture plate and pipette 300 microliters of previously prepared calcium phosphate solution into each well.
Then, cover the plate with the lid and incubate the plate at 37 degrees Celsius for three days. After three days, aspirate the pre-calcification solution from the pre-calcified 96 well cell culture plates and add 300 microliters of calcium phosphate solution to each well. Cover the plates with a lid and incubate at 37 degrees Celsius for one day.
Then invert the plate to pour out the solution and thoroughly wash the plate thrice with deionized water. Immediately dry the plate using a hair dryer or compressed carbon dioxide or nitrogen gas to maintain a uniform surface. Now, irradiate the coated plate on a clean bench with ultraviolet rays for one hour.
Use the coated plate immediately or seal the plate with Parafilm and store it at room temperature. Dilute 15 milliliters of fresh blood with an equal volume of PBS and mix them by inverting or pipetting several times. Then take 15 milliliters of density gradient solution in a 50 milliliter conical tube, tilt the tube, and carefully layer 30 milliliters of diluted blood sample on the 15 milliliters of density gradient solution.
Later, centrifuge the tube in a swinging bucket rotor without break at 810 times G for 20 minutes at 20 degrees Celsius. Then aspirate the upper layer leaving the mononuclear cell layer undisturbed at the interface. Carefully transfer the mononuclear cell layer to a new 50 milliliter conical tube.
Fill the tube with PBS, mix, and centrifuge at 300 times G for 10 minutes at 20 degrees Celsius. Re-suspend PBMCs in a complete alpha-MEM containing 20 nanograms per milliliters macrophage colony stimulating factor, and seed 2.5 times 10 to the fifth cells per centimeter square PBMCs in a flask. Incubate the calcium phosphate coated plates with 50 microliters of fetal bovine serum at 37 degrees Celsius for one hour.
Next, wash the flask twice with PBS to detach the osteoclast precursors from dead or non-adherent cells, then add four milliliters of trypsin per 75 square centimeter flask. After 30 minutes, stop the digestion by adding four milliliters of complete alpha-MEM, then carefully detach the cells using a cell scraper. Transfer the cells into a 50 milliliter tube and count the total number of cells using a counting chamber.
Centrifuge the tube at 350 times G for seven minutes to pellet the cells and resusspend the pellet and complete alpha MEM containing M-CSF and RANKL to get one times 10 to the six cells per milliliter. Aspirate the fetal bovine serum from the calcium phosphate coated plate, and pipette 200 microliters of cell suspension per well. After the incubation, wash the cells twice with PBS.
Fix the cells with 4%paraformaldehyde for 10 minutes and wash again with PBS. For staining, add permeabilization buffer to the fixed cells and incubate them for five minutes. To stain the actin filaments, incubate the cells with 100 microliters of Alexa Fluor 546 labeled phalloidin solution in PBS for 30 minutes, then aspirate the staining solution and stain the nuclei using 100 microliters of Hoechst for 10 minutes.
Stain calcium phosphate coating with 100 microliters of 10 micromolar calcein and PBS for 10 minutes. After washing thrice with PBS, capture images. For Von Kossa staining of calcium phosphate coating add 50 microliters of 5%silver nitrate and deionized water to each well, and incubate the plate under ultraviolet radiation for one hour until the coating on the bottom of the wells has turned brown.
The calcium phosphate coating at the bottom of the 96 well plates appeared to be uniform and well adhered. After nine days of culture in M-CSF and RANKL on the calcium phosphate coated plates, osteoclast precursors formed resorption pits and large osteoclasts. The multi-nucleated osteoclasts located in the pits expressed high TRAP levels.
The mature osteoclasts showed the presence of three or more nuclei and the distinct actin ring. The green calcium phosphate coating showed the presence of black resorption pits. The pit area fluorescence images was measured using the thresholding tool of image J.The number and size of osteoclasts were calculated using ROI manager and the polygon selection tool of image J.Osteoclasts'precursors grown in the absence of RANKL could not form resorption pits, however M-CSF and RANKL facilitated the formation of osteoclastogenic pits.
And the pit area was quantified using image J.Immediately drying the coated plate with airflow helps to make a uniform calcium phosphate coating. Otherwise it's prone to form a thicker coating in the middle of the well.
