Resorption Pit Assay: An In Vitro Technique to Quantify Calcium Resorption Activity of Mature Osteoclasts using Calcium Phosphate-Coated Culture Plates
Transcription
Resuspend PBMCs in a complete alpha-MEM containing 20 ng/mL macrophage colony-stimulating factor, and seed 2.5 x 105 cells/cm2 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 4 milliliters of trypsin per 75 cm2 flask. After 30 minutes, stop the digestion by adding 4 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 x g for seven minutes to pellet the cells, and resuspend the pellet in complete alpha-MEM containing M-CSF and RANKL to get 1 x 106 cells/mL.
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 AlexaFluor 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 in PBS for 10 minutes. After washing thrice with PBS, capture images.
