Peroxidase conjugated wheat germ agglutinin

Bone marrow was flushed from the long bones of 6–8 week old C57BL/6 WT or DGKζ^−/− mice and cultured in α-minimum Eagle’s medium (α-MEM; Sigma-Aldrich, St. Louis, MO) containing 5% heat-inactivated fetal bovine serum, glutamine and 1/50 volume CMG 14–12 culture supernatant as source of M-CSF (17 ) for 3 days to generate BMMs. For osteoclastogenesis, BMMs were further cultured in the presence of 100 ng/ml GST-RANKL (purified from BL21 with pGEX-6-RANKL plasmid, using HOOK GST protein purification kit, G-BIOSCIENCES, St. Louis, MO) and 25 ng/ml M-CSF (Sigma-Aldrich, St. Louis, MO) for an additional 3–5 days. For TRAP staining, cells were fixed in 4% paraformaldehyde and stained using the leukocyte acid phosphatase kit (Sigma-Aldrich). For bone resorption, BMMs were cultured on tissue culture plates in the presence of osteoclastogenic medium for 2 days, lifted, and replated in equal number on bone slices for additional 2 days. Cells were scraped and bones stained with 20 μg/ml peroxidase-conjugated wheat-germ agglutinin (Sigma-Aldrich) for 1h at room temperature, followed by incubation with 3,3′-diaminobenzidine (Sigma-Aldrich) for 30 min. Bone resorptive pits were analyzed using a light microscope (Nikon) and quantified using Image J software.

After 6 days of culture, bone slices were incubated in 0.5 N NaOH for 30 seconds and cells scraped off using a cotton swab, then incubated with 20 μg/mL peroxidase-conjugated wheat germ agglutinin (Sigma) in PBS for 30 min, washed with PBS three times, and exposed to 3,3′-Diaminobenzidine tablets (Sigma; D4168) for 15 min before washing. BioQuant OSTEO 2010 (BioQuant Image Analysis Corporation, Nashville, TN, USA) was used to quantify pit area.

Retrovirally transduced and selected BMMs were cultured on bovine bone slices for 6 days in 1:50 dilution of CMG 14-12 cell supernatant and 30ng/ml GST-RANKL, replaced with fresh media and cytokines every 24 hrs. For pit assay, bone slices were incubated in 0.5N NaOH for 30 sec and cells scraped off using a cotton swab, then incubated with 20 μg/ml peroxidase-conjugated wheat germ agglutinin in PBS (Sigma) for 30 min, washed with PBS 3 times, and exposed to 3,3′-diaminobenzidine (Sigma) for 15 min before washing and drying. BioQuant OSTEO 2010 (BioQuant Image Analysis Corporation) was used to quantify pit area. For assessment of CTX release in culture, media was collected for the final 24 h of culture, between days 6 and 7 and analyzed using the CrossLaps for Culture CTX-I ELISA kit (Immunodiagnosticsystems AC-07F1) per manufacturer instructions.