Recombinant mouse macrophage colony stimulating factor m csf

Dulbecco's Modified Eagle Medium (DMEM, low glucose), RPMI1640, DPBS and fluorescein isothiocyanate-dextran molecular weight 70,000 (FDx70) were purchased from Life Technologies. Recombinant mouse macrophage-colony stimulating factor (M-CSF) was from R&D Systems. Phorbol 12-myristate 13-acetate (PMA), U73122 and rottlerin were from Abcam. Calphostin C was from Calbiochem. Farnesyl thiosalicylic acid (FTS) was from Santa Cruz. MK-2206 was from Apexbio.

Minimum essential medium-α (MEM-α), fetal bovine serum (FBS), and penicillin–streptomycin (P/S) were purchased from Life Technologies (Carlsbad, CA, USA). Recombinant mouse macrophage colony-stimulating factor (M-CSF) and enzyme-linked immunosorbent assay (ELISA) kits for interleukin-1β (IL-1β) were obtained from R&D Systems (Minneapolis, MN, USA). An ELISA kit for immunoglobulin E (IgE), interleukin-6 (IL-6), interleukin-18 (IL-18), and thymic stromal lymphopoietin (TSLP) was supplied by Invitrogen (Carlsbad, CA, USA). Lipopolysaccharide from Escherichia coli O26:B6 (LPS), adenosine 5′-triphosphate disodium salt hydrate (ATP) and 2,4-dinitrochloro benzene (DNCB) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Radioimmunoprecipitation assay buffer (RIPA buffer) and skim milk powder were supplied by ELPIS Biotech (Deajeon, Republic of Korea) and BD Difco (Sparks, MD, USA), respectively. All other materials and reagents used in the study were of the highest quality available.

Minimum essential medium alpha medium (α-MEM), RPMI 1640 medium, Dulbecco’s modified Eagle’s medium (DMEM)/F-12 phenol red-free medium (1:1), fetal bovine serum (FBS), antibiotic-antimycotic mixture (100×), phosphate-buffered saline (PBS), and 0.25% trypsin-EDTA (1×) were purchased from Gibco BRL Co. (Grand Island, NY). Dimethyl sulfoxide (DMSO), LPS, and 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St Louis, MO). Recombinant mouse soluble RANK ligand (sRANKL) was purchased from Koma Biotech (Seoul, Republic of Korea). Recombinant mouse macrophage colony-stimulating factor (M-CSF) was purchased from R&D System (Minneapolis, MN). EETC was provided by COSMAX Inc. R&I Center (Seongnam City, Republic of Korea).

Recombinant murine IL-1β and TNF were obtained from PeproTech (Rocky Hill, NJ, USA). Recombinant mouse macrophage colony-stimulating factor (M-CSF) was purchased from R&D Systems® (Minneapolis, MN, USA). Lipopolysaccharide (LPS), Pam3Csk4, and Poly(I:C) were purchased from InvivoGen (San Diego, CA, USA). Antibodies to c-Myc (9E10), TRADD (H278), ERK2 (D2) and β-tubulin (H235) were obtained from Santa Cruz Biotechnology®, Inc (Dallas, TX, USA). Antibodies specific for phospho-JNK/SAPK (Thr183/Tyr185), phospho-p44/42 MAPK (pERK) and phospho-IκB (Ser32/36) (5A5) were purchased from Cell Signalling Technology® (Danvers, MA, USA). Antibodies to Na⁺-K⁺ ATPase (ab69312) and green fluorescent protein (GFP; ab7671) were purchased from Abcam® (Cambridge, UK). Antibody to GAPDH was obtained from Sigma-Aldrich® (St. Louis, MO, USA).

Recombinant mouse macrophage-colony stimulating factor (M-CSF) and RANKL were purchased from R&D Systems, USA. Fetal Bovine Serum (FBS) was obtained from Hyclone Laboratories. Alpha-minimal essential medium (α-MEM) and Penicillin Streptomycin were acquired from Gibco. MBMs were isolated from the femurs of 8-week-old C57BL/6 mice and cultured as previously described 27 -29 . Firstly, collected MBM was cultured in α-MEM with 10% FBS containing 20 ng/ml M-CSF overnight. Adherent cell was primary monocytes. Non-adherent MBM was collected and seeded into 12-well plates (1×10⁵cell/well) or 24-well plates (5×10⁴cell/well), in wells or in bovine cortical bone slices, according to the experiment requirements. Then cells were cultured in α-MEM with 10% FBS containing M-CSF (10ng/mL) and RANKL (10ng/mL) for 5 days or 7 days continuously to induce osteoclasts.

Saikosaponins A, B₁, B₂, B₃, B₄, C, and D isolated from B. falcatum L. were provided by Professor Kim [23 (link), 24 (link)]. The saikosaponins were dissolved in dimethyl sulfoxide (DMSO) and then diluted with culture medium just before use. Minimum essential medium alpha (α-MEM), Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), Dulbecco's phosphate buffered saline (PBS), antibiotic-antimycotic mixture, and 0.25% trypsin-EDTA were purchased from Gibco BRL (Grand Island, NY). Histopaque-1083, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO). Recombinant mouse RANKL was purchased from Koma Biotech (Seoul, Republic of Korea) and recombinant mouse macrophage-colony stimulating factor (M-CSF) was obtained from R&D system (Minneapolis, MN). TGF-β was purchased from PeproTech (Rocky Hill, NJ). All reagents used in this study were of analytical grade.