The bone and skeletal muscle tissues were collected from wild-type male and CS mice described above [9 (link),12 (link)]. Briefly, the muscles were incubated with Dulbecco’s modified Eagle’s medium (DMEM+) solution (EuroClone S.p.A., Milano, Italy) composed of 1X antibiotics (1%), L-glutamine (1%), FBS (10%) (EuroClone, S.p.A., Milano, Italy) and enriched with collagenase type IX (0.1%) (SIGMA Chemical Co., Milan, Italy). The fibers were then enzymatically isolated from the Flexor digitorum brevis (FDB) mice muscles in the shaker incubator for 2 h [9 (link),30 (link),31 (link)].
The culture of primary bone cells from adult mice was obtained from long bone such as femora or calvaria, as previously described [9 (link),31 (link)] Bones were collected, cleaned, and flushed to remove the internal bone marrow cells. Small bone pieces of around 1 mm3 were treated with trypsin-EDTA 0.25% (w/v) for 1 h and with 0.2% collagenase solution for an additional hour in a shaking water bath to remove all remaining soft tissue and adherent cells. Clean bone pieces have been cultured in a basal medium enriched with 50 µg/mL of ascorbic acid. As previously described, osteoblasts from calvaria chips were positive for Alizarin red staining and positive for PCR gene expression on cell pellets [32 (link)].
The culture of primary bone cells from adult mice was obtained from long bone such as femora or calvaria, as previously described [9 (link),31 (link)] Bones were collected, cleaned, and flushed to remove the internal bone marrow cells. Small bone pieces of around 1 mm3 were treated with trypsin-EDTA 0.25% (w/v) for 1 h and with 0.2% collagenase solution for an additional hour in a shaking water bath to remove all remaining soft tissue and adherent cells. Clean bone pieces have been cultured in a basal medium enriched with 50 µg/mL of ascorbic acid. As previously described, osteoblasts from calvaria chips were positive for Alizarin red staining and positive for PCR gene expression on cell pellets [32 (link)].
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