To analyze sufficient cell numbers in downstream experiments, rib cage growth plates from newborn and 1-mo-old mice were incubated in collagenase II solution (450 U/ml; Biochrom) for 6 h at 37°C. Femoral growth plates could not be used due to the insufficient yield in cell numbers, especially from 1-mo-old mice. To obtain adequate numbers of cells, rib cage–derived chondrocytes from two to three animals of both sexes were pooled unless otherwise stated (Fig. 7 B ) and cultured in DMEM-F12 supplemented with 10% FCS (Biochrom), penicillin (100 U/ml; Biochrom), streptomycin (100 µg/ml; Biochrom), ascorbate (44 µg/ml; Sigma-Aldrich), and L-ascorbate-2-phosphate (130 µg/ml; Sigma-Aldrich). After 24 h, chondrocytes were detached with collagenase II in DMEM-F12 (2 mg/ml; Biochrom), centrifuged, and resuspended in medium. Cells were cultured for another 3 d and then used for experiments. The pH of the cell culture supernatant was determined using a Seven-Easy-pH-Meter (Mettler).
Collagenase 2 solution
Manufactured by Harvard Bioscience
Sourced in Germany
Collagenase II solution is a purified enzyme designed for use in cell isolation and tissue dissociation applications. It is effective for the digestion of collagen, a primary structural component of the extracellular matrix.
Automatically generated - may contain errors
Lab products found in correlation
Fetal calf serum (fcs), by Harvard Bioscience (1 mentions)
Dmem f12, by Harvard Bioscience (1 mentions)
Ascorbate, by Merck Group (1 mentions)
L ascorbate 2 phosphate, by Merck Group (1 mentions)
Penicillin, by Harvard Bioscience (1 mentions)
Streptomycin, by Harvard Bioscience (1 mentions)
Seveneasy ph meter, by Mettler Toledo (1 mentions)
2 protocols using collagenase 2 solution
1
Isolation and Culture of Chondrocytes
2
Osteogenic Differentiation of Ad-MSCs
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Fat tissue (4–6 g per sample) was cut into mm-sized pieces using a scalpel and washed three to four times with phosphate buffered saline (PBS). For digestion, the pieces were incubated in 0.7% collagenase II solution (Biochrom, Berlin, Germany) for about 30 min at 37 °C. Collagenase digestion was stopped by adding culture medium (DMEM 4.5 g/L glucose, 10% FCS). After centrifugation at 600× g for 10 min, the Ad-MSC pellet was re-suspended in culture medium and expanded to passage 3 (37 °C, 5% CO2). Flow cytometry revealed that in passage 3, Ad-MSCs were negative for CD14, CD45, and HLA-DR and positive for CD73, CD90, and CD105 (Figure 6 ), which is in line with our earlier reports [44 (link)]. Thus, in passage 3, the cells (104 cells/cm2) were osteogenically differentiated for 14 days. The osteogenic differentiation medium (DMEM 4.5 g/L glucose, 1% FCS, 200 μM L-ascorbic acid 2-phosphate, 10 mM β-glycerol phosphate, 25 mM HEPES, 1.5 mM CaCl2, 5 μM cholecalciferol) was replaced every 3–4 days [12 ].
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