Primary murine bone marrow stromal cells (BMSC) were differentiated to osteogenic cells using standard osteogenic medium in DMEM (Invitrogen, Darmstadt, Germany) for 7 days.(8 (link),12 (link)–14 (link)) Before treatment, cells were switched to DMEM containing 1% FCS overnight and then treated with 1 μg/mL LPS (Sigma-Aldrich) or 50 ng/mL TNF-α (R&D Systems, Frankfurt, Germany) for 48 hours. In some experiments, BMSC were isolated from TLR2/4 KO mice (C57BL/6 backgroud). To block TNF-α actions, cells were pretreated for 2 hours with 1 μg/mL of soluble TNF receptors (TNFR1 and TNFR2, both from R&D Systems).
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Osteogenic Differentiation of Murine BMSC
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Corresponding Organization :
Other organizations : Heinrich Heine University Düsseldorf, Universitätsmedizin Greifswald, National Cancer Institute, Center for Cancer Research
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Variable analysis
independent variables
- LPS (1 μg/mL)
- TNF-α (50 ng/mL)
- Knockout of TLR2/4 in BMSC
- Pretreatment with soluble TNF receptors (1 μg/mL TNFR1 and TNFR2)
- Osteogenic differentiation of BMSC
- Primary murine bone marrow stromal cells (BMSC)
- Standard osteogenic medium in DMEM for 7 days
- Cells switched to DMEM containing 1% FCS overnight before treatment
- Positive control: Osteogenic differentiation of BMSC using standard osteogenic medium
- Negative control: BMSC from TLR2/4 KO mice
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