Cathepsin k activity assay

A total of 5 × 10⁵ BMDMs were seeded per well in a six-well plate. Cells were then stimulated with either PMT or M-CSF or M-CSF+sRANKL for 6 days, and cathepsin K Activity Assay was performed (Abcam). Cells were lysed in 200 µl of cathepsin K cell lysis buffer and incubated on ice for 10 min. Cell debris were centrifuged at 14,000 g for 5 min, and the supernatants were removed. The amount of protein in the lysates was determined with a BCA Assay (Pierce), and the amount was adjusted to 3 µg of protein in 50 µl of lysis buffer per well of a 96-well plate. Fifty microliters of cathepsin K reaction buffer was added to each well. The Ac-LR-AFC substrate was added to a final concentration of 140 µM, and the plate was incubated for 2 h at 37°C. Fluorescence was measured with a microplate reader (FLUOstar OPTIMA; BMG LABTECH) with an excitation of 355 nm and an emission of 520 nm.

RAW264.7 cells were seeded at 1 × 10⁵ cells per well in a 6-well plate. Cells were pre-treated with rapamycin or DMSO for 1 h before cells were stimulated with PMT or left untreated. After 4 days of differentiation, a cathepsin K Activity Assay was performed (Abcam). Cells were lysed in 50 μl of cathepsin K cell lysis buffer and incubated on ice for 10 min. Cell debris were centrifuged at 15,000 rpm for 5 min and the supernatants were removed. The amount of protein in the lysates was determined with a BCA Assay (Pierce) and the amount of protein for each assay was adjusted to 10 μg of protein in 50 μl of lysis buffer per well of a 96-well plate. 50 μl of cathepsin K reaction buffer was added to each well. The Ac-LR-AFC substrate was added to a final concentration of 200 μM and the plate was incubated for 1 h at 37 °C. Fluorescence was measured with a microplate-reader (FLUOstar OPTIMA; BMG LABTECH) with an excitation of 355 nm and an emission of 520 nm.