Recombinant murine m csf

For harvesting bone marrow, mice were anesthetized by intraperitoneal (i.p.) injection of midazolam (8 mg/kg, Roche Diagnostics), medetomidine (0.4 mg/kg, Orion) and fentanyl (0.08 mg/kg, Janssen Pharmaceutica). Mice were sacrificed by cervical dislocation.
Bone marrow derived monocytes were isolated and differentiated as described previously [42] (link). In brief, bone marrow suspensions were isolated from WT and RP105^−/− mice by flushing the femurs and tibias with PBS. Cells were cultured for 5 days in RPMI medium supplemented with 10% fetal calf serum, 2 mmol/L l-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin and 20 ng/mL recombinant murine M-CSF (eBioscience) to generate BM-derived monocytes. After 5 days, non-adherent cells were harvested and analyzed by FACS for CD11b and Ly6C expression in order to determine monocyte purity (∼84%).

Murine macrophages were differentiated in vitro as previously described [19 (link)]. In brief, 8–12 week old iRhom2 knockout (KO) or wild-type (WT) mice were sacrificed by carbon dioxide (CO₂) inhalation, and femurs and tibia collected. After removing joints from the bones, femurs and tibia were flushed with 2 ml RPMI (Gibco, part of Thermo Fischer Scientific, Billings, Montana, United States). Cells were strained through a nylon mesh to remove debris and clumps, into a 15 ml centrifuge tube. Then, cells were resuspended in 15 ml complete RPMI, and centrifuged at 230 × g for 5 min. Cell pellet was resuspended in 1 ml of red blood cell lysis buffer (Sigma-Aldrich, St. Louis, Missouri, United States) for 3 min to remove erythrocytes, then diluted in 15 ml RPMI and centrifuged again at 230 × g for 5 min. Pellet was resuspended in RPMI, then 10⁶ cells were plated in a bacteriological Petri Dish with 10 ml complete RPMI supplemented with 100 ng/ml recombinant murine M-CSF (Peprotech, London, UK). After 1 week, cells were differentiated into macrophages (98% of cells were F4/80 positive–not shown) and then used for further analysis (secretome analysis and FACS).

BMDMs and BMDCs were generated as described [28 (link)]. The bone marrow cells (1×10⁷) were cultured in RPMI medium 1640 containing 10% FBS and 10 ng/mL recombinant murine M-CSF (Peprotech) or GM-CSF-containing conditional medium in a 100-mm dish for 5 or 9 days for generation of BMDMs or BMDCs respectively.

Baicalin was purchased from Sigma‐Aldrich (St. Louis, MO, USA). Recombinant murine M‐CSF and RANKL were purchased from Peprotech (Rocky Hill, NJ, USA). Anti‐Mitf, anti‐ERK, anti‐phospho ERK, anti‐p38, anti‐phospho p38 and anti‐β‐actin antibodies were all purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti‐MMP9 antibody was purchased from Abcam, Inc. (dilution 1:1000; Danvers, MA, USA). α‐Modified essential medium (α‐MEM) and rhodamine phalloidin were obtained from Life Technologies Corp. (Carlsbad, CA, USA), and TRAP staining kit was purchased from Sigma‐Aldrich. Mounting medium was purchased from Vector Laboratories, Inc. (Burlingame, CA, USA). Cell Counting Kit‐8 was purchased from Dojindo Molecular Technologies (Dojindo, Tokyo, Japan). All other chemicals were obtained from Sigma‐Aldrich.

Mouse bone marrow cells were isolated from 10–12-week-old FXR^+/+ or FXR^−/− C57BL/6J background mice. Bone marrow cells were cultured in complete α-MEM containing 10% (v/v) fetal bovine serum (FBS), supplemented with 5 ng/ml recombinant murine M-CSF (PeproTech) for 12 h to separate adherent and non-adherent cells. The non-adherent cells were then harvested and cultured with 30 ng/ml M-CSF. After 4 days of culturing, the floating cells were removed and the attached cells were used as BMMs. All cells were cultured in α-MEM containing 5% (v/v) FBS at 37°C in a humidified atmosphere in 5% CO₂ conditions.

In vitro osteoclast cultures were performed essentially as described before (54 (link), 55 (link)). Bone marrow cells obtained by flushing the tibia and femur of wild type or mutant mice were cultured in the presence of 10 ng/ml murine M-CSF (Peprotech) for 2 days in α-MEM medium (Sigma) supplemented with 10% FCS (Gibco) and antibiotics. Non-adherent cells were then plated at the concentration of 1.5 × 10⁵ cells/cm² and cultured in the presence of 50 ng/ml recombinant murine M-CSF and 50 ng/ml murine RANKL (Peprotech) with medium changes every 2 days. In parallel macrophage cultures, the cells were cultured under identical conditions except that RANKL was omitted.
Cultures were terminated and osteoclast-specific staining was performed using a commercial tartrate-resistant acid phosphatase (TRAP) staining kit (Sigma-Aldrich) at the indicated times after the first addition of RANKL. Photomicrographs were taken using a Leica DMI6000B inverted microscope. The images were then analyzed either manually or by the ImageJ software. Osteoclasts were defined as TRAP-positive cells with 3 or more nuclei.
For in vitro resorption assays, osteoclasts were cultured under similar conditions for 7 days on an artificial hydroxyapatite surface (Sigma-Aldrich) followed by washing, imaging by dark field microscopy and further analysis by ImageJ software.