Osteoclastogenesis Assay with LPS and MTT

LPS from E. Coli serotype 055:B5 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Aldrich (St. Louis, MO, United States). As TRAP staining reagent, Acid phosphatase Kit 387-A was used (Sigma-Aldrich, St. Louis, MO, United States). TRAP reaction buffer was prepared as described by the manufacturer’s instructions (Sigma Aldrich, St. Louis, MO, United States). Recombinant murine RANKL was from Peprotech (PeproTech EC, Ltd., London, United Kingdom). pSrc/Src ratio was assessed by immunoblot with rabbit Phospho-Src Family (Tyr416) Antibody #2101 (Cell Signaling Technology, Danvers, MA, USA), recognizing the phosphorylation at tyrosine 424 in murine c-Src, followed by the detection of total Src by rabbit monoclonal antibody 36D10 (Cell Signaling Technology, Danvers, MA, United States), as previously shown (Manni et al., 2020 (link)).