Rankl 462 tec

Cell‐culture supernatants of osteoblasts, left unstimulated or stimulated for 4 h with C5a and/or Pam3, were applied on osteoclast cultures, to investigate the osteoclastogenic potential. RAW 264.7 (ATCC^® TIB‐71) cells were seeded at a density of 1500 cells/cm² in 96‐well plates and differentiated into osteoclast‐like cells. Osteoblast supernatants were added to the osteoclast medium (DMEM (ATCC, Manassas, USA), supplemented with 10% heat‐inactivated FCS, 1% penicillin/streptomycin and 1% L‐glutamine (all from Gibco)) in a 1:1 ratio and with a final concentration of 10 ng/ml RANKL (462‐TEC, R&D Systems, Wiesbaden, Germany) and 5 ng/ml human recombinant M‐CSF (Merck). Additionally, PMX‐53 (1.1 μg/ml), MyD88 inhibitor ST 2825 (10 μM), and a CXCL10‐antibody (10 μg/ml, R&D Systems) were applied together with the osteoblast supernatant. In addition, control groups were included, where C5a (100 ng/ml), Pam3 (100 ng/ml) or recombinant mouse CXCL10 (100 ng/ml, PeproTech, Hamburg, Germany), with or without CXCL10‐antibody, were added to the osteoclast medium without osteoblast supernatant. Cells were kept at 37°C under 5% CO₂. Osteoclastogenic differentiation was assessed after 5 days by counting multinucleated (>2 nuclei) tartrate‐resistant acid phosphatase (TRAP)‐positive cells using light microscopy. Additionally, mRNA samples were obtained.

Bone marrow from tibia and femur of WT and cKO mice was flushed with α-MEM and cells pelleted by centrifugation at 1500 rpm for 5 min. Bone marrow cells were then cultured in complete α-MEM media supplemented with 10% fetal bovine serum, 1% antibiotic, and 30 ng/ml of recombinant mouse macrophage colony stimulating factor (MCSF, 416-ML, R&D systems, Minneapolis, MN, USA) at 37 °C in a humidified 5% CO₂ atmosphere to allow cell attachment. After 2 days, non-adherent cells were removed and discarded; adherent cells were cultured to confluence and treated as bone marrow macrophages (BMMs). BMMs of passage 1 or 2 were used in the experiments below. BMMs were seeded on to 96-well plate (6 × 10³ cells/well) and treated with 30 ng/ml MCSF, and 100/25 ng/ml RANKL (462-TEC, R&D systems) to induce OC formation. The media was replaced with supplemented MCSF and RANKL (with and without BS) every 2 days and after 7 days of culture, the cells were fixed in 2.5% glutaraldehyde and stained with Acid Phosphatase staining kit (387 A, Sigma–Aldrich, St. Louis, MO, USA) according to the protocol of the manufacturer. TRAP-positive multinucleated cells with more than three nuclei were counted as OCs using light microscopy. The number of OCs in each well was counted using ImageJ software.