Anti mil 6

Cells from buffy coat were grown in α‐MEM supplemented with 10% FCS containing 20 ng/ml of macrophage colony‐stimulating factor (M‐CSF), feeding with fresh media until they reach a suitable confluency. Briefly, 2 × 10⁴ human osteoclasts from buffy coat were seeded into wells of a 96‐well plate, supplemented with α‐MEM containing 20 ng/ml M‐CSF plus 20 ng/ml of receptor activator of nuclear factor kB ligand (RANKL). As a control, cells were incubated without RANKL were included. The following day, cells were fed with fresh α‐MEM containing M‐CSF and RANKL as appropriate, plus the accurate treatments; 20% of each MLO‐Y4 cell‐conditioned medium. Moreover, in some experiments, neutralizing antibody anti‐mCXCL5 (Thermo Fisher Scientific) and anti‐mIL‐6 (R&D Systems) were used. After 3 days, the medium was removed, cells were rinsed off with PBS to eliminate nonadherent cells. Cells were fixed with 4% p‐formaldehyde, permeabilized with 100% methanol for 20 min and stained with hematoxylin for 5 min. The differentiation of human monocytic cells into osteoclasts were determined by morphology, observing the formation of giant cells with at least three or more nuclei.

Migration assay was performed using Costar transwell cell culture chamber inserts (Corning Costar Corporation) with an 8 µm pore size. Briefly, 5 × 10⁴ RAW 264.7 cells or human osteoclasts from buffy coat were placed in the upper chamber in 200 µl of DMEM supplemented with 10% FBS or α‐MEM supplemented with 10% FCS, respectively. Standard medium with 1% FBS or FCS and containing 20% of each MLO‐Y4 cell‐conditioned medium for 18 h were placed in the lower compartment. Furthermore, in others experiments, 2 µg/ml of neutralizing antibody anti‐mCXCL5 (Thermo Fisher Scientific) and 1 µg/ml of anti‐mIL‐6 (R&D Systems) were used. Then, the medium and the upper cell layer were removed with the aid of a cotton swab, and cells on the underside of the transwell were fixed with PFA 4%, stained with crystal violet (Sigma Aldrich) and counted in five randomly selected fields at X200 magnification using Leica DM 5500B microscope.