Calvarial Bone Resorption Assay

Calvariae from 5-d-old wild-type or DKO mice were isolated aseptically, cleaned, and cultured for 16 h at 37°C in 0.5 ml of BGJb medium (Life Technologies) containing 1 mg/ml BSA (fraction V; Sigma-Aldrich; Moxon et al., 2015 (link)). Half calvariae were transferred to fresh medium with 0.1 μM parathyroid hormone (H-4835.0005; Bachem) in the presence or absence of an anti–galectin-3 monoclonal antibody (sc-32790L; Santa Cruz) or an anti-Lrp1 monoclonal antibody (MA1-27198; Thermo Fisher Scientific), and cultured for an additional 5 d. Culture supernatant was collected for bone resorption marker CTX-I level detection using RatLaps CTX-I EIA kit (AC-06F1; Immunodiagnostic Systems). The half calvariae were either fixed for immunostaining with an anti-TRAP polyclonal antibody (sc-30833; Santa Cruz), an anti–galectin-3 monoclonal antibody (#125401; Biolegend; clone M3/38), or an anti-V-type proton pump-3 (Vpp3) polyclonal antibody (ab200839; Abcam) as describe above or snap-frozen for TRAP activity assay.

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Zhu L., Tang Y., Li X.Y., Kerk S.A., Lyssiotis C.A., Sun X., Wang Z., Cho J.S., Ma J, & Weiss S.J. (2023). Proteolytic regulation of a galectin-3/Lrp1 axis controls osteoclast-mediated bone resorption. The Journal of Cell Biology, 222(4), e202206121.

Publication 2023

BGJb medium BSA sc-32790L MA1-27198 RatLaps CTX-I EIA kit sc-30833 ab200839

Anti antibody Antibody Assay Bone resorption Calvariae Clone Frozen Galectin 3 Immunodiagnostic Mice Monoclonal antibody Moxon Parathyroid hormone Proton

Corresponding Organization : Wuhan University

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Variable analysis

independent variables

Mouse genotype (wild-type or DKO)
Parathyroid hormone (0.1 μM) treatment
Anti-galectin-3 monoclonal antibody treatment
Anti-Lrp1 monoclonal antibody treatment

dependent variables

Bone resorption marker (CTX-I) level in culture supernatant
TRAP-positive osteoclast staining
TRAP activity

control variables

Culture duration (5 days)
Culture medium (BGJb with 1 mg/ml BSA)
Culture temperature (37°C)

controls

Positive control: Parathyroid hormone treatment
Negative control: No parathyroid hormone treatment

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