Whole bone marrow cells were obtain

Whole bone marrow cells were obtained from the femora and tibiae of C57BL/6J mice (10-week-old, male) according to the protocol approved by the Animal Experimental Committees of Showa University (code number: 17050, 04/01/2017). Cells (1 × 105 cells per cm2) were incubated in α-MEM (Gibco) containing 10% FBS supplemented with 4000 U/mL Leukoprol (M-CSF: macrophage colony stimulating factor, JCR Pharmaceuticals Co. Ltd., Hyogo, Japan) for 2 days to induce bone marrow macrophages (BMM). BMM were further incubated with 50 ng/mL of soluble receptor activator of NF-κB ligand (sRANKL, Wako Pure Chemical Industries Ltd.) in the presence of Leukoprol (4000 U/mL) for 3 days to differentiate into maturated OC. BMM were incubated in the presence of 0–111 μg/mL MBE during only the first 2 days with Leukoprol, then for 3 days with Leukoprol and sRANKL, or throughout the culture period. The culture medium was changed every second day. OC differentiation was examined by measuring the intensity of tartrate-resistant acid phosphatase (TRAP) staining at 540 nm using a microplate reader (Infinite 200, Tecan Japan Co. Ltd., Kawasaki, Japan). qPCR was performed with a StepOne Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA) using SYBR Green (Toyobo Co. Ltd., Osaka, Japan). The sequence of the specific primers for the qPCR is listed in Table 1. The level of mRNA expression was normalized with that of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) expression. Cell proliferation of BMM was examined by 5-bromo-2′-deoxyuridine (BrdU) incorporation assay. Briefly, bone marrow cells (3 × 105 cells per cm2) were cultured in α-MEM containing 10% FBS supplemented with 4000 U/mL Leukoprol for 2 days and incubated for 4 h in the presence of BrdU. BrdU incorporation was detected based on a colorimetric ELISA assay kit (Cell Proliferation ELISA, BrdU; Roche, Basal, Switzerland).

結果 (繁體中文) 2:

根據秀華大學動物實驗委員會批准的協定（代碼：17050，04/01/2017），從C57BL/6J小鼠（10周大，雄性）的親骨細胞獲得整個骨髓細胞。細胞（每cm21×105細胞）在β-MEM（Gibco）中孵育，含有10%FBS，輔以4000U/mL Leukoprol（M-CSF：巨噬細胞群落刺激因數，日本兵庫JCR製藥有限公司），誘導骨髓巨噬細胞（BMM）。BMM進一步孵育，使用NF-βB配體（sRANKL， Wako純化學工業有限公司）在Leukoprol（4000 U/mL）的存在下3天，以分化成成熟的OC。 BMM在僅與Leukoprol的2天內，在0~111μg/mL MBE的存在下孵育，然後與Leukoprol和sRANKL一起孵育3天，或在整個培養期。文化媒介每第二天改變一次。使用微孔板閱讀器（無限200，日本川崎Tecan日本株式會社）測量540nm層耐酸磷酸酶（TRAP）染色的強度，從而對OC分化進行了研究。qPCR使用StepOne即時PCR系統（美國麻塞諸塞州沃爾瑟姆的賽默費舍爾科學系統）使用SYBR Green（日本大阪東洋博株式會社）執行。表 1 列出了 qPCR 特定引注的順序。mRNA表達水準與甘油醛-3-磷酸脫氫酶（Gapdh）表達水準歸一化。BMM的細胞增殖通過5-溴-2+-去氧尿氨酸（BrdU）結合測定。簡單地說，骨髓細胞（每cm23×105細胞）在含有10%FBS的β-MEM中培養，輔以4000U/mL Leukoprol2天，並在BrdU存在的情況下孵育4小時。根據色度ELISA測定試劑盒（細胞增殖ELISA，BrdU;羅氏，巴塞爾，瑞士）。

結果 (繁體中文) 3:

根據昭和大學動物實驗委員會準予的方案（程式碼：17050，04/01/2017），從C57BL/6J小鼠（10周齡，雄性）的股骨和脛骨獲得全骨髓細胞。細胞（每平方釐米1×105個細胞）在含有10%胎牛血清的α-MEM（Gibco）中培養2天以誘導骨髓巨噬細胞（BMM）。BMM與50ng/mL的NF-κB配體可溶性受體啟動劑（sRANKL，Wako Pure Chemical Industries Ltd.）在白細胞介素（4000u/mL）存在下進一步孵育3天，以分化為成熟OC。BMM在0-111μg/mL MBE存在下僅在最初的2天內與白血病醇一起孵育，然後在3天內與白血病醇和sRANKL一起孵育，或在整個培養期內孵育。培養基每天更換一次。通過使用微型平板閱讀器（無限200，日本川崎帝肯株式會社）在540 nm處量測抗酒石酸酸性磷酸酶（TRAP）染色强度來檢測OC分化。qPCR是使用SYBR-Green（日本大阪豐田有限公司）的step1實时PCR系統（Thermo Fisher Scientific，Waltham，MA，USA）進行的。錶1列出了qPCR特异性引子的序列。mRNA表達水准與甘油醛-3-磷酸脫氫酶（Gapdh）表達水准呈正相關。用5-溴脫氧尿苷（BrdU）摻入法檢測骨髓基質細胞增殖。簡單地說，骨髓細胞（3×105個/cm~2）在含有10%胎牛血清的α-MEM中培養2天，並在BrdU存在下培養4小時。基於比色ELISA試劑盒（細胞增殖ELISA，BrdU；羅氏，基礎，瑞士）檢測BrdU摻入。