Phenol red free alpha mem

Primary bone marrow macrophages were harvested from the femurs and tibiae of 4-week-old WT and Smad1^fl/fl/Smad5^fl/fl; C-fms Cre cKO mice as previously described [4 (link)]. Briefly, femurs and tibiae were dissected and adherent tissue was removed. The ends of the bones were cut and the marrow was flushed from the inner compartments. Red blood cells were lysed from the flushed bone marrow tissue with RBC lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA, pH7.4) and the remaining cells were plated and cultured overnight in 100 mm tissue culture dishes (TPP, MidSci) in osteoclast media (phenol red-free alpha-MEM (Gibco) with 5% fetal bovine serum (Hyclone), 25 units/mL penicillin/streptomycin (Invitrogen), 400 mM L-Glutamine (Invitrogen), and supplemented with 1% CMG 14–12 supernatant (culture supernatant containing M-CSF). CMG14-12 cells were obtained from Dr. Sunao Takeshita (Nagoya City University, Nagoya, Japan). The non-adherent cell population, including osteoclast precursor cells, was then separated and re-plated in 12-well plates (TPP, MidSci) at 2x10⁶ cells/cm² in osteoclast media supplemented with 1% CMG 14–12 culture supernatant. Two days later cells were refed with 1% CMG 14–12 culture supernatant and 30 ng/mL RANKL (R&D Systems) to stimulate osteoclast differentiation. Cultures were fed every other day for up to 4 days.

Primary bone marrow macrophages were harvested from the femurs and tibiae of 4-week-old WT, HET and KO mice. The femurs and tibiae were dissected and adherent tissue was removed. The ends of the bones were cut and the marrow was flushed from the inner compartments. Red blood cells were lysed from the flushed bone marrow tissue with RBC lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA, pH7.4) and the remaining cells were plated and cultured overnight in 100 mm tissue culture dishes (Corning) in osteoclast media (phenol red-free alpha-MEM (Gibco) with 5% heat-inactivated fetal bovine serum (Hyclone), 25 units/mL penicillin/streptomycin (Invitrogen), 400 mM L-Glutamine (Invitrogen), and supplemented with 1% CMG 14–12 culture supernatant[11 (link)] containing 1.2 μg/mL M-CSF). The non-adherent cell population, including osteoclast precursor cells, was then separated and re-plated in 24-well plates (Corning) at 1.7x10⁴ cells/cm² in osteoclast media supplemented with 1% CMG 14–12 culture supernatant. Two days later cells were refed with 1% CMG 14–12 culture supernatant and 30 ng/mL RANKL (R&D Systems) to stimulate osteoclast differentiation. Osteoclast activity was assayed on calcium phosphate plates (Corning). The demineralized area was photographed by light microscopy and then analyzed using NIH Image J.

Primary bone marrow macrophages (BMMs) were isolated from the femora and tibiae of C57BL/6 mice. The femora and tibiae were dissected out and adherent tissue was removed. The epiphyses of these long bones were removed, and the bone marrow was flushed from the diaphysis. Red blood cells were lysed from the flushed marrow using red blood cell lysis buffer (150 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA, pH 7.4), and the resulting cells were plated and cultured overnight in 100 mm tissue culture dishes (MidSci) in osteoclast media (phenol red-free alpha-MEM [Gibco] with 5% fetal bovine serum [Hyclone], 25 units/mL penicillin/streptomycin [Invitrogen], 400 mM L-Glutamine [Invitrogen], and supplemented with 1% CMG 14–12 culture supernatant containing M-CSF). CMG 14–12 cell line was obtained from Dr. Sunao Takeshita (Nagoya City University, Nagoya, Japan) [27 (link)]. The non-adherent cell population was then re-plated in 12-well plates (MidSci) at 2 x 10⁵ cells/well in osteoclast media supplemented with 1% CMG 14–12 culture supernatant for 48 hours. Cells were then fed every two days with osteoclast media containing 1% CMG culture supernatant plus 10 ng/mL RANKL (R&D Systems) to stimulate osteoclastogenesis.