mined optimal concentrations of IC for human MC mediator release was 8.8 g/ml antibody with 0.13 g/ml NP-BSA (not shown).
Human fibroblasts (PC37303A1-S-Passage 1) from the synovium 
of RA patients (n = 5) having joint replacement Epetraborole (hydrochloride) surgery (Asterand, Detroit, Michigan, USA) were plated in triplicate on a 96 well plate using RPMI complete medium. Cells were incubated overnight with or without fullerene derivatives at various concentrations, washed, and activated with or without 10 ng/ml of TNF- for 12 hours. The supernatants (in triplicate) were assayed for IL-6 and IL-8 using ELISA [36].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 40magnification 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).
Human MC were plated in triplicate in 96-well plates and incubated as above with fullerene derivatives, 10205015 washed, and activated with or without FcR stimulation for 24 hours as above. After treatment, cells were fixed by adding formalin to a final concentration of 3.7% formaldehyde and incubated at room temperature for 10 minutes. After fixation, the plates were washed with PBS then permeabilized (PBS containing 0.1% Triton-X-100) by washing three times for 10 minutes on a bench top shaker and finally rinsed once with PBS containing 0.1% Tween 20. Cells were blocked using 50 L/well blocking buffer (Li-Cor Biosciences, Odyssey Blocking Buffer, Lincoln, Nebraska, USA) for one hour at room temperature. Primary antibodies, Actin (1:1000) (Cell Signaling Technology, Danvers, Massachusetts, USA) and NF-B (1:500) (Cell Signaling Technology, Danvers, Massachusetts, USA), incubated overnight at 4 using 20 L/well, washed three times with 100 L/well PBS-Tween, and centrifuged at room temperature. Secondary antibodies were prepared as for Western blotting with a few modifications: IRDye 680CW and 800 CW conjugates of goat-anti-mouse-IgG (Li-Cor Biosciences, Lincoln, Nebraska, USA) were used at 1:1000 dilution for detection of antibody targets in the 800 and 700 nm channels (green and red respectively). Plates were incubated with 20 L/well secondary antibody solutions for 1.5 hours at room temperature in the dark, washed three times for 10 minutes with PBS-Tween at room temperature, and filled with 50 L/well of PBS to reduce surface disturbances when scanning. The 800-channel antibody signals were normalized to the 700-channel signals derived from IR-680 conjugated secondary. Background control wells were prepared by omitting primary antibodies, IRDye 680CW and IRDye 800CW (ie secondary only, Li-Cor Biosciences, Lincoln, Nebraska, USA). Plates were scanned and analyzed using an O
