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Fig. 5. Kinetics of dextran endocytosis. For uptake kinetics, osteoclasts plated on dentine were incubated for 1 minute, 5 minutes, 10 minutes and 30 minutes with TRITC-labelled dextran-3000 before fixation and immunodecoration with antibodies against the _vß₃ integrin to identify the cells (A-C). To follow the trafficking of the label through the osteoclast, cells plated on dentine were labelled for 10 minutes with TRITC-labelled dextran-3000 before a chase period of 15 minutes, 30 minutes or 60 minutes in medium without label (D,E). Computer-assisted 3D reconstruction of x-y sections taken at a 0.5 µm step size was used to visualize the trafficking of the dextran through the osteoclasts (A,B,D, red indicates TRITC-labelled dextran and blue indicates _vß₃ integrin). At early time points, the dextran is exclusively found close to the bone surface and the pit (A,B) but, after 60 minutes of chase, the label is distributed throughout the cell and can be observed close to the basolateral membrane surface and outside the cell (D, arrowhead). The amount of fluorescence inside of the cells was determined using the Leica TCS-NT software package and expressed as arbitrary fluorescence units. Columns show mean fluorescence and s.e.m. (C,E).

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