Generation of highly active human osteoclasts from peripheral blood mononuclear cells: a requirement for TGF-β1. Human osteoclasts were generated from peripheral blood mononuclear cells (PBMNC) as described under Materials and Methods. The medium contained either a mix of 10% FCS, dexamethasone (1 μM), M-CSF (25 ng/ml) and RANKL (50 ng/ml) (- TGF-β1) or this mix with 5 ng/ml TGF-β1 (+ TGF-β1). Number of TRAP-positive multinucleated cells and area of resorbed bovine bone or dentin was quantified microscopically. Upper panel: Quantification of TRAP-positive multinucleated cell number and pit area. The results are shown as means ± SEM; n is a number of independent experiments. Middle panel: Microphotographs of TRAP-stained cells on plastic and of toluidine blue-stained pits on dentin. Bottom panel: Molecular characterization of cells during osteoclast differentiation from PBMNC. Total RNA was extracted at the indicated times and expression of 8 osteoclast markers and 18S rRNA was measured by radioactive quantitative RT-PCR. RT-: reverse transcription omitted. OSM: osteoclast stimulating medium, containing the above mix of serum, dexamethasone, M-CSF, RANKL, and TGF-β1. No OSM cultures contained medium and FCS only. B: bone slices; D: dentin slices. Nd: not determined.