Osteoclastic Cathepsin K Evaluation

Osteoclastic culture medium was supplemented with alendronate or risedronate (10⁻⁵ M) from 0 to 72 h, and a control group was treated with osteoclastic medium without BP stimulation in the presence of 2 brefeldins (BFAs) for four hours. Then, the levels of cathepsin K were evaluated by flow cytometry according to a previous report with modifications [30 (link)]. The cellular analysis was performed on a BD FACSCalibur platform (Becton Dickinson, San José, CA), with a total of 5,000 events collected with CellQuest software (Becton Dickinson). Cells were harvested from the culture plate using 0.25% trypsin (Gibco) and centrifuged at 1,500 rpm for 5 minutes. The pelleted cells were washed twice with 1 ml of 1x PBS containing 0.8% bovine serum albumin (BSA) and 0.02% sodium azide. Fixation and permeabilization were performed with an intracellular staining kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer's specifications. Osteoclastic cells were washed in 1 ml of 1x PBS plus BSA. A primary rabbit antihuman cathepsin k polyclonal antibody was incubated with the cells for 30–45 minutes at room temperature; we added a secondary rabbit anti-IgG antibody coupled to phycoerythrin isothiocyanate (PE) for 30–45 minutes at room temperature. A control sample was generated following the same steps for intracellular staining (Figure 4).