Differentiation of CD11c⁺ splenic DCs into functional OCLs in vitro. (A) Splenic DCs sorted from normal mice were cultured for 8 days either without cytokines or with 25 ng/mL M-CSF and 30 ng/mL RANK-L allowing their differentiation into multinucleated cells. (B) These differentiated cells were analyzed for their TRAP activity. The presence of the a3 protein (rhodamine) and the calcitonin receptor (fluorescein isothiocyanate) was also analyzed by immunofluorescence. Incubation with a preimmune serum served as negative control. Nuclei were stained with DAPI. (C) Resorption activity was assessed on dentine slices for cells cultured without cytokines or with M-CSF and RANK-L. Arrows indicate the presence of resorption lacunae with cytokine-treated cells. Equivalent results were found with DCs sorted from Balb/c and C57BL/6 mice. (D) Sorted splenic DCs were cultured for 8 days without cytokines or with various combinations of M-CSF (25 ng/mL), RANK-L (30 ng/mL), and GM-CSF (10 ng/mL). TRAP activity was analyzed and TRAP⁺ multinucleated cells (MNCs) were counted. The results are the mean plus or minus SD of 4 equivalent wells. (E) The kinetics of Nfatc1 expression was analyzed by real-time RT-PCR between days 0 and 4 of culture on cells treated with M-CSF (M) and RANK-L (R) and with our without GM-CSF (GM). The results are the mean plus or minus SD of 4 equivalent wells. All results are representative of 3 independent experiments. Images were captured using (panels A,D) an Axiovert 200 microscope (Carl Zeiss, Oberkochen, Germany) with a 10×/0.25 Ph1 or a 20×/0.3 Ph1 Var1 Plan objective (Carl Zeiss) and a JVC KY-F50 camera, (panel B) an Axioskop microscope (Carl Zeiss) with a 40×/1.30 oil objective (Carl Zeiss), an AxioCam HRc camera (Carl Zeiss), and Gel/Mount medium (Biomeda, Foster City, CA), and (panel C) an Axioskop microscope (Zeiss) with 20×/0.75 Fluar or 40×/0.75 Plan-Neofluar objectives. All images were acquired using AxioVision 4.5 software (Zeiss).