Osteogenesis differentiation medium
Osteogenesis differentiation medium is a cell culture medium designed to support the differentiation of cells towards the osteogenic lineage. It provides the necessary components to promote the formation of bone-like tissues in vitro.
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9 protocols using osteogenesis differentiation medium
Osteogenic Differentiation of iNCMSCs
Multilineage Differentiation of Mouse MSCs
To evaluate their multipotent differentiation potential, MSCs were incubated at passage 5 in a 12‐well plate at a density of 5 × 103 cells/cm2 with osteogenesis differentiation medium (Gibco) or at a density of 1 × 104 cells/cm2 with an adipogenesis differentiation kit (Gibco) for 3 weeks. According to the manufacturer's instructions, other MSCs were seeded at a higher density of 1 × 107 cells per milliliter to formulate a micromass and cultured with a STEMPRO chondrogenesis differentiation kit (Gibco) with medium changes every 3 days for 2 weeks. Osteoblasts were stained using Alizarin Red S (Sigma), adipocytes were stained with Oil Red O (Sigma), and chondrogenesis pellets were stained with Toluidine Blue (Sigma).
Evaluating Mesenchymal Stem Cell Potency
Differentiation and Doxorubicin Cytotoxicity
Cellular growth media of DMEM (Dulbecco’s modified Eagle’s medium; 1 gram per liter of D-glucose), adipogenesis differentiation medium, and osteogenesis differentiation medium were purchased form Gibco Company (USA) and were then supplemented with penicillin antibiotic (50 units per mL), fungicide amphotericin B (2.5 µg per mL), essential amino acids, and L-glutamine. Doxorubicin drug was purchased from Sigma Company. A solution of 1000 μM in phosphate buffer saline (PBS) was prepared, and the stock solution was kept at -20 oC. Alizarin Red, Oil Red O, and propidium iodide (PI) were obtained from Sigma Company (USA). Fetal calf serum (FCS) and trypsin/EDTA (0.5%) were purchased from Gibco Company (USA), and collagen was supplied from STEM CELL Technologies (Canada). Monoclonal antibodies against surface markers were also provided from Dako Company (Denmark). The measurement kit of cytotoxicity detection for measuring lactate dehydrogenase (LDH) activity was purchased from Roche (Germany), and the solvents were supplied from Merk (Germany).
ASC Osteogenic Differentiation Assay
Quantitative Analysis of Stem Cell Differentiation
For the chondrogenesis assay, pellets were prepared by spinning down 1 × 105 cells and incubating in a 15-mL conical tube in chondrogenesis differentiation medium (Invitrogen) with the medium changed every 3 days. After 28 days of culturing, cells were fixed with 4% formaldehyde, and stained with Alcian Blue (Merck, Darmstadt, Germany).
Directed Differentiation of Mesenchymal Stem Cells
Adipogenic and Osteogenic Differentiation
Multilineage Differentiation of Canine ADSCs
For osteogenic differentiation, passage 2 cADSC were seeded on 12-well plates at a concentration 2 × 105 cells/well and incubated in DMEM supplemented with 10% FBS and 1% antibiotic-antimycotic solution for 24 h. The medium was then replaced with 1 mL of osteogenesis differentiation medium (Thermo Fisher Scientific) and changed every 3 days. For osteogenic analysis, mineral deposits were analyzed quantitatively by Alizarin Red staining after 21 days.
For chondrogenic differentiation, 2 × 105 passage 2 cADSC were suspended in 1 mL of chondrogenic differentiation medium (STEMCELL Technologies, Vancouver, BC, Canada) and 0.5 mL was aliquoted into a 15 mL conical tube and then centrifuged at 300× g for 5 min. The lid was then loosened and the cells were incubated for 3 days, after which 0.5 mL of medium was added and replaced every 3 days. Chondrogenic differentiation was evaluated by Alcian Blue staining after 21 days.
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