Crosslaps for culture ctx 1 elisa

Osteoclasts cultured on plastic were stained using the Tartrate Resistant Acid Phosphatase (TRAP) Kit (Sigma-Aldrich), following the manufacturer's instructions. Osteoclasts differentiated on bone slices were stained using alendronate conjugated to Alexa Fluor 488,^{24 (link)} TRITC-conjugated phalloidin (Sigma-Aldrich) and TO-PRO-3 (Thermo Fisher Scientific). To evaluate bone resorption, the same bone slices were used for toluidine blue staining as previously described.^{25 (link)} TRAP and toluidine blue images were acquired on a Zeiss AxioImager M2m microscope, while immunofluorescence staining images were acquired on a Leica TCS SP5 Laser Scanning Confocal microscope.
Quantification of CTX-I was performed on culture supernatants using CrossLaps for Culture (CTX-I) ELISA (Immunodiagnostic Systems), according to the manufacturer’s instructions.

BAL cells were collected d14 post i.t. saline or silica challenge and plated on plastic or bovine bone slices (Immunodiagnostic Systems, UK) in 96-well plates (2.0 x 10⁴ cells/well) in α-minimum essential medium (α-MEM; Gibco) containing 10% FBS in the presence of various concentrations (0-100 ng/ml) of recombinant murine RANKL (PeproTech) and 1:40 dilution of CMG12-14 (murine M-CSF producing cell line (69 (link))) conditioned medium (equivalent to 30 ng/ml of recombinant human M-CSF). Medium was changed on day 2, 4, and 5. Cells plated on plastic were fixed in formalin and stained for TRAP activity after 6 days in culture. For actin ring staining, cells on bone slices were fixed on day 6 in formalin and permeabilized in 1% Triton X-100, rinsed with PBS, and stained with AlexaFluor 488-phalloidin (Invitogen) (70 (link)). For the bone pitting assay, cells on bone slices were removed by scrubbing the bone surface with toothbrush, and resorption pits were visualized by incubation with 20 μg/ml peroxidase-conjugated wheat germ agglutinin and staining with 3,3’-diaminobenzidine (Sigma-Aldrich) (69 (link), 70 (link)). The levels of CTX-I in culture medium over bone slices were measured using the CrossLaps^® for culture (CTX-I) ELISA (Immunodiagnostic Systems) (71 (link)).