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9 protocols using adipogenesis differentiation medium

1

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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2

Adipogenesis Induction in Mesenchymal Stem Cells

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iNCMSCs were seeded at a density of 1 × 105/cm2 and cultured in 10%FBS-aMEM until over confluent. Subsequently, the cells were cultured in adipogenesis differentiation medium (Gibco) for 72 hours, followed by incubation in adipogenic maintenance medium (10%FBS-aMEM, 10 μg/mL insulin) for 72 hours. Adipogenesis was induced by cycles of induction/maintenance [54 (link)]. Cells cultured in 10%FBS-aMEM served as a control. To detect the formation of lipid vacuoles, cells were fixed with 4% PFA, washed, and stained with oil red O solution (0.5% oil red O (Sigma-Aldrich) in 60% isopropanol) for 15 minutes. The stained dye was eluted with 100% isopropanol, and the absorbance was measured at 520 nm.
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3

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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4

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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5

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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6

Adipogenic Differentiation Assay Protocol

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OASCs during passages three to five were seeded at a density of 2.5 × 104 cells per cm2 in 24-well PL plates in DMEM with 10% fetal bovine serum. At 90% confluence on day 3, the medium was switched to the Adipogenesis Differentiation Medium (Invitrogen, Carlsbad, CA). Torin 2 (0.02 µM), PX12 (10 µM), withaferin A (0.5 µM), isoliquiritigenin (25 µM), mitoxantrone (0.025 µM), and MLN 8054 (5 µM) were added 3 days after differentiation induction of OASC into mature adipocytes. After 21 days of culturing, the cells were fixed with 4% paraformaldehyde and stained with Oil Red O for adipocytes from the Adipogenesis Assay Kit (Cayman Chemical Company, Ann Arbor, MI) according to the manufacturer's protocol. Cells with oil droplets stained by Oil Red O were quantified via spectrophotometry at an absorbance of OD 490 nm in triplicate cultures (Fig. 1).
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7

Bimatoprost Enhances Adipocyte Differentiation

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OASCs during passages three to five were seeded at a density of 2.5 × 104 cells per cm2 in 24-well PL plates in DMEM with 10% FBS. At 90% confluence on day 3, the medium was switched to the Adipogenesis Differentiation Medium (Invitrogen, Carlsbad, CA, USA). Bimatoprost (1 μM; Selleckchem Catalog No.S1407) was added on day 6 after differentiation of OASC into mature adipocytes. After 21 days of culturing, the cells were fixed with 4% paraformaldehyde (PFA) and stained with oil red O for adipocytes from the Adipogenesis Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA) according to the manufacturer's protocol. Cells with oil droplets stained by oil red O were quantified via spectrophotometry at an absorbance of OD 492 nm in triplicate cultures.
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8

Multilineage Differentiation of Progenitor Cells

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PSC were seeded in 12-well plates, grown to 50–60% confluence, and incubated in the
StemPro™ osteogenesis differentiation medium, adipogenesis differentiation medium and
chondrogenesis differentiation medium (Thermo Fisher Scientific), respectively. After
14–21 days induction, cells were fixed using 4% paraformaldehyde for 30 min. For
adipogenic differentiation, cells were stained with 2% Oil Red O for 10 min. For
osteogenic differentiation, cells were stained with 1% Alizarin Red S for 3 min. For
chondrogenic differentiation, cells were stained with 1% Alcian Blue solution prepared in
0.1 N HCl for 30 min. The images were visualized under a bright-field microscope.
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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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