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Osteogenesis differentiation medium

Manufactured by Thermo Fisher Scientific
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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

1

Osteogenic Differentiation of iNCMSCs

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iNCMSCs were seeded at a density of 5 × 104/cm2 and cultured in osteogenesis differentiation medium (Gibco). Cells cultured in 10%FBS-aMEM served as a control. Differentiation was confirmed by assessing alkaline phosphatase (ALP) activity and calcium deposition. For analysis, cells were rinsed twice with PBS, fixed with 4% paraformaldehyde (PFA) for 10 minutes, and washed with water. Alkaline phosphatase (ALP) activity was detected using the BCIP/NBT Color Development Substrate (Promega, Wisconsin, USA), according to the manufacturer's instruction. Calcium deposition was detected by 1% alizarin red S solution (pH 6.4) (Muto Pure Chemicals, Tokyo, Japan) for 10 minutes at room temperature and then washed thoroughly with water. For von Kossa staining, culture wells were stained with 5% silver nitrate (Wako) under ambient light for 1 hour and then fixed with 5% sodium thiosulfate (Wako) for 5 min to remove nonreacted silver.
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2

Multilineage Differentiation of Mouse MSCs

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Balb/c mice (2–3 weeks old) were decapitated, and the femur and tibia were carefully removed. Bone marrow cells were flushed with a syringe (27‐gauge) inserted into one end of the bone and cultured at a density of 3 × 105 cell/cm2 with low‐glucose Dulbecco's modified Eagle's medium (DMEM; Gibco) containing 15% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Gibco). After 2 hours, nonadherent cells were removed via medium changes, which were repeated every 3 days until the cultured cells reached 70%–80% confluence. Cells were then digested with TrypLE Express (Gibco) for 2 minutes and passaged. All experiments used MSCs at passage 5–8.
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).
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3

Evaluating Mesenchymal Stem Cell Potency

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To evaluate MSC abilities, adipogenic and osteogenic differentiation assays were performed on isolated cells. Osteogenesis differentiation medium (Gibco) or adipogenesis differentiation medium (Gibco) was added into a culture when the fusion rate reached approximately 80%. The cells were cultured at 37°C in 5% (vol/vol) CO2 in 100% humidified air. The media were changed every 3 days, and the cells were cultured for 2 to 3 weeks before collection. Then, Alizarin Red S staining was used to analyze osteogenic lineages, whereas Oil Red O was used to analyze lipid droplets. Adipogenic and osteogenic differentiation assays were conducted three times for all four donor cells.
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4

Differentiation and Doxorubicin Cytotoxicity

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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).
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5

ASC Osteogenic Differentiation Assay

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To assess the differentiation capacity of ASC towards osteoblasts, ASC (passage 1) were seeded in 12-well culture dishes at 10,000 cells/cm2. The cells were then cultured in monolayer in Osteogenesis Differentiation Medium (Gibco). Control cells were cultured in normal culture medium. Both differentiation and control media were changed twice a week. Differentiation was stopped at 12 days by fixating the cells with 4 % formaldehyde at RT for 10 min. Differentiation was demonstrated by staining alkaline phosphatase (ALP). For this, cells were incubated in 0.2 M TRIS-hydrochloride (pH 10), 0.2 M calcium, 0.1 M magnesium chloride solution for 10 min, whereafter a solution containing 0.2 M TRIS-hydrochloride (pH 10), 0.2 M calcium chloride, 0.1 M magnesium chloride solution and nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) was added (1:100, Roche Diagnostics, Mannheim, Germany) for 15 min. This stains ALP blue. Microscopic images were taken using a Zeiss microscope.
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6

Quantitative Analysis of Stem Cell Differentiation

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For assays of adipogenesis or osteogenesis, expanded single cells during passages three to five were seeded at the density of 2.5 × 104 cells/cm2 in 24-well plates in DMEM with 10% FBS. At 90% confluence, the medium was switched to the adipogenesis differentiation medium or the osteogenesis differentiation medium, respectively (Invitrogen) and changed every 3 days. After 21 days of culturing, cells were fixed with 4% formaldehyde and stained with oil red O for adipocytes by adipogenesis Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA) or with 2% Alizarin Red for osteocytes following the manufacturer's protocol. Cells with oil droplet stained by Oil Red were quantified by measuring at OD at 492 nm in triplicate cultures. Mineralized cells with positive Alizarin Red staining (Alfa Aesar, Tewksbury, MA, USA) were quantified by measuring OD at 405 nm in triplicate cultures.
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).
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7

Directed Differentiation of Mesenchymal Stem Cells

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For differentiation into keratocytes, 3 × 105 cells were spun down for pellet preparation and cultured in the Keratocyte Differentiation Medium consisting of Advanced DMEM with 10 ng/ml fibroblast growth factor 2 and 0.5 mM ascorbic acid; Thermo Fisher Scientific) for 21 days44 (link). For differentiation into corneal endothelial cells, single cells were seeded at the density of 1 × 104 cells per cm2 in 24-well plate coated with collagen IV in MESCM + 5% FBS. After cell adhesion in 24 h, the medium was switched to Corneal Endothelial Differentiation Medium consisting of low-glucose DMEM with 10% FBS and 50 µg/ml gentamicin, and 1.25 µg/ml amphotericin B) for 28 days45 . For differentiation into adipocytes or osteocytes, single cells were seeded at the density of 1 × 104 cells per cm2 in 24-well plate coated with Matrigel in MESCM + 5% FBS. After cell adhesion in 24 h, the medium was switched to the Adipogenesis Differentiation Medium (Invitrogen) or Osteogenesis Differentiation Medium (Invitrogen), respectively, for 21 days18 (link). For differentiation into chondrocytes, 3 × 105 cells were prepared for pellets and cultured in Chondrogenesis Differentiation Medium (Life Line) for 28 days18 (link). All the above media were changed every 3 days.
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8

Adipogenic and Osteogenic Differentiation

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MSCs were grown to 90% confluency and cultured for 28 days in StemPro Adipogenesis or Osteogenesis differentiation medium (Thermo Fisher, USA). Adipogenic and osteogenic terminal differentiation was monitored by Oil Red O (Sigma Aldrich, USA) or Alizarine Red S (Sigma Aldrich, USA) staining, respectively.
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9

Multilineage Differentiation of Canine ADSCs

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For adipogenic differentiation, passage 2 cADSC were seeded on 12-well plates at a concentration 4 × 104 cells/well and cultured in DMEM supplemented with 10% FBS and 1% antibiotic-antimycotic solution until 80% confluency. The medium was then replaced with 1 mL of adipogenesis differentiation medium (Thermo Fisher Scientific) and changed every 3 days. Adipogenesis was analyzed by Oil Red O staining after 21 days.
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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