Leukocyte acid phosphatase assay

Osteoclasts were obtained from peripheral blood monocytes (PBMC) as described previously [11 (link)]. Fresh buffy coats (AVIS, Bologna, Italy) were diluted with PBS, layered over Hystopaque (Sigma, St. Louis, MO), and centrifuged at 900g for 30 minutes. The mononuclear cells were extracted from the interphase of the PBS and Hystopaque and centrifuged at 400g for 5 minutes. Cells were rinsed in PBS and seeded on tissue-culture glass or plastic ware in D-MEM supplemented with 10% FCS and incubated at 37°C in a humidified 5% CO₂ atmosphere. Cells were seeded at the density of 3,000,000/cm². After 1 hour, medium was discarded and replaced with conditioned medium from confluent cultures of WT- or R527H-transfected U2-OS cells (25% conditioned medium and 75% normal medium). Conditioned medium was replaced every 4 days for 14 days. In order to verify the differentiation of mononuclear cells to osteoclasts, after 8 days of culture, cells were analyzed for tartrate resistant acid phosphatase (TRACP) activity by cytochemistry (Acid Phosphatase Leukocyte assay, Sigma), and stained with Hoechst 33258 (1.25 g/ml). TRACP-positive cells containing 3 or more nuclei were considered as differentiated osteoclasts.

Human osteoclasts were obtained from peripheral blood mononuclear cells (PBMC) as described previously [37 ]. Mononuclear cells were cultured in complete DMEM containing RANKL (25 ng/ml) and M-CSF (25 ng/ml). Fullerene derivatives were added at varying concentrations and remained in the medium until the end of the experiment (eight days). Cells were washed, cytocentrifuge preparations made, and analyzed for tartrate resistant acid phosphatase (TRAP) activity by cytochemistry (Sigma Aldrich, Acid Phosphatase Leukocyte assay, St. Louis, Missouri, USA). At eight days multinucleated cells containing three or more nuclei were counted. TRAP-positive cells containing three or more nuclei were considered to be differentiated osteoclasts. Experiments were done in triplicate, 25 microscopy fields at 40× magnification were evaluated for each sample. Quantitation of osteoclasts TRAP staining was performed using the infrared imaging analysis (Li-Cor Biosciences, Odyssey Imaging CLx System, Lincoln, Nebraska, USA).