Rankl

Osteoclast precursors were generated ex vivo as described previously [18 (link), 26 ]. Briefly, after flushing and harvesting bone marrow, cells were seeded into tissue culture plates and allowed to grow overnight. Subsequently, non-adherent cells were transferred to non-treated suspension plates with 35 ng/mL MCSF (Biolegend, 574804) and allowed to grow up to 70–80% confluence. Cells were later harvested using Accutase cell disassociation reagent (MilliporeSigma, A6964) and seeded at a density of 26,000 cells per cm² in 10% α-MEM media (MilliporeSigma, M0894) supplemented with 35 ng/mL MCSF and 100 ng/mL RANKL (Shenandoah Biotechnology, 200-04-100μg). After, a period of 4 days, multinuclear cells were visible as a result of osteoclastogeneis and TRAP stained following the guidelines mentioned in the Leucocyte acid phosphatase or TRAP kit (Sigma Aldrich, 387-A). In case of LPS (ultrapure, Invivogen, tlrl-3pelps) treated groups, cells were treated with MCSF + RANKL + LPS or initially stimulated with MCSF + RANKL for a period of 48 hr and later treated with MCSF and LPS (100 ng/mL) for another 72 hr before TRAP staining or other functional assessments as described previously [16 (link)].