Cultures were incubated with alpha minimum essential medium conta

Cultures were incubated with alpha minimum essential medium containing 10% fetal calf serum, 100 U mL penicillin, 100g mL streptomycin, and 100M CLO or ALN in the absence or presence of 100 ng mL recombinant human RANKL. After 48 hours, the culture all targets media were removed and adherent cells were fixed with 4% formaldehyde Inhibitors,Modulators,Libraries and either stained with 1g mL 4,6 diamidino 2 phenylindole in PBS or stained for TRAP. The number of TRAP positive multinucleated osteoclasts per well or the proportion of osteoclasts with DAPI stained nuclei showing characteristic apoptotic nuclear morphology was determined using a Zeiss Axiovert 135 microscope and �� 20 objective. Analysis of osteoclast morphology by scanning electron microscopy Bone marrow cells from rabbit long bones were seeded onto discs of elephant ivory in 96 well plates and cultured with MEM containing 10% FCS with 50M CLO or ALN in the absence or presence of 100 ng mL recombinant human RANKL.

After 24 hours, cells were fixed in 2. 5% Inhibitors,Modulators,Libraries glutaraldehyde and 2. 5 mM MgCl2 in 0. 089 M phosphate buffer for 3 hours at room temperature. Discs were washed overnight in 0. 1 M phosphate buffer, post fixed in osmium tetroxide for 1 hour, washed in distilled water, and dehydrated through a graded series of ethanol solutions. The samples were critical point dried from CO2, glued onto aluminium stubs with colloidal silver adhesive, sputter coated with 20 nm platinum, Inhibitors,Modulators,Libraries and examined in a Jeol JSM 35CF scan ning electron microscope oper ating at 10 kV.

Quantification of osteoclast mediated bone resorption Rabbit bone marrow cells Inhibitors,Modulators,Libraries were seeded onto ivory discs as described above and cultured with MEM containing 10% FCS with 100M CLO or ALN in the absence or presence of 100 ng mL recombinant human RANKL. After 48 hours, the media were removed, cells were wiped from the ivory discs, and the total area of min eral resorbed per disc was determined using a reflected light microscope. Measurement of caspase 9 activity in osteoclasts Rabbit osteoclasts, purified as described above, were cul tured with MEM containing 100M ALN 100 ng mL RANKL for 48 hours. Unfixed, adherent cells were stained using an Apofluor Green Caspase Activity Assay kit. This involves the covalent binding of a fluorescently labelled, cell permeable caspase inhibitor to active caspase 9, thus allowing the detec tion of cells with caspase 9 activity.

Cells were counterstained with Hoechst 33342, washed to remove excess stain, and vis ualised using a Zeiss Axiovert 135 microscope and �� 20 Inhibitors,Modulators,Libraries objective. Western blot analysis Mature osteoclasts were isolated from rabbit long bones, seeded into 10 cm diameter Petri dishes, and purified merely as pre viously described. Purified osteoclasts were cultured for 48 hours with 100 ng mL RANKL or with 100M ALN 100 ng mL RANKL.

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