Lung-derived prominin-1.
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Exposure of prominin-1+ cells to M-CSF for 7 days resulted in formation of E.coli phagocytising F4/80-positive cells .
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Osteoblast, osteoclast and macrophage differentiation.
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Bone marrow derived monocytes were seeded in 24-well culture plates and induced to differentiate into macrophages or osteoclasts via the addition of M-CSF or M-CSF + RANKL respectively.
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Cell activation assay and immunodetection of cytokines
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Single spleen cell suspensions were prepared by mechanical disruption on a cell strainer (70 µm pore diameter , , ) in Dulbecco's D-PBS .
- Red blood cells were lysed using Hemolytic Gey's Solution as previously described
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Osteoclastogenesis assays
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Osteoclastogenesis assays were performed as in 5 cells were plated in each well of a 24-well dish with 60 ng/mL RANKL for 3 days.
- After 3 days, 2×103 MDA-231, shControl, or shEGFR-MDA-231 cells were plated with the bone marrow cells, with 60 ng/mL RANKL and 10 ng/mL MCSF and grown for 3 days before TRAP staining.
- For TRAP staining, cells were washed with 1×PBS, fixed with ice cold methanol for 10 minutes, and stained in fresh TRAP solution for 15 minutes at 37°C.
- TRAP solution was replaced with 1×PBS for cell counting under the microscope.
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PLCγ2 is required for in vitro osteoclast development.
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(a) Representative TRAP-stained images of wild-type and PLCγ2−/− bone marrow cells cultured in the presence of the indicated concentrations of recombinant murine M-CSF and RANKL for 4 days.
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Generation of conditional TAK1-deficient mice.
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Colony formation by BM cells from Map3k7
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Generation of bone marrow-derived macrophages
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Bone marrow-derived macrophages (BMDM) were generated by flushing the bone marrow from the femurs of BALB/c mice with RPMI 1640 media.
- Freshly collected bone marrow cells were cultured overnight in complete media (CM; RPMI 1640, 10% fetal bovine serum [Atlanta, ], 10 mM HEPES buffer, 10 mM nonessential amino acids, 10 mM sodium pyruvate) containing 5 ng/ml recombinant murine M-CSF .
- The non-adherent cells were then collected and cultured for an additional six days in CM with 30 ng/ml M-CSF to generate macrophages.
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Pro-resorptive cytokine expression and osteoclastogenesis in the synovial fluid of patients with gouty arthritis.
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PBMCs, SFMCs, and T cell-depleted SFMCs (3 × 105 cells/well) were cultured for 10 days in the presence of macrophage colony-stimulating factor (M-CSF; 30 ng/ml) at the different RANKL concentrations and then were stained for tartrate-resistant acid phosphatase .
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TcREG inhibit osteoclast activity in an antigen- and contact-independent manner by secreted cytokines.
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Cells were re-fed with medium containing M-CSF and RANKL every 2 to 3 days.
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Induction of 2nd iPSCs
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All isolated somatic cells were cultured in the presence of Dox (2 ug/ml) for induction of 2nd iPSCs.
- Fibroblasts and hematopoietic cells were cultured in ES/iPSC medium.
- FLCD45 were cultured in the presence of 10 ng/ml human TPO, 10 ng/ml mouse EPO, 10 ng/ml mouse IL-3, 10 ng/ml mouse IL-6, 10 ng/ml mouse Flt3 ligand, 10 ng/ml mouse GM-CSF, 10 ng/ml mouse VEGF and 50 ng/ml mouse SCF .
- HSCs, HPCs and MPs were cultured in the presence of 10 ng/ml human TPO, 10 ng/ml mouse IL-3, 10 ng/ml mouse IL-6 and 10 ng/ml mouse Flt3 ligand .
- Macrophages were cultured in the presence of 5 ng/ml M-CSF .
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