hUC-MSCs at passage 3 were seeded in six-well plates in complete medium in triplicate at a final density of 5000 cells per cm2 according to the previous report with some modifications [30 (link)] Forty-eight hours later, designated as day 0, differentiation was initiated using the adipogenic induction medium (AdvanceSTEM adipogenic differentiation medium supplemented with 10% AdvanceSTEM stem cell growth supplement, Thermo Scientific, Rockford, IL), according to the manufacturer's instructions. The medium was changed every 3-4 days, and the experiment was terminated after 3 weeks. The differentiated hUC-MSCs were fixed with 4% paraformaldehyde (PFA) and stained with oil-red-O (IHC World, Woodstock, MD) to visualize cytoplasmic lipid-rich vacuoles.
Advancestem stem cell growth supplement
Manufactured by Thermo Fisher Scientific
AdvanceSTEM is a stem cell growth supplement developed by Thermo Fisher Scientific. It is designed to support the in vitro expansion and maintenance of human pluripotent stem cells.
Automatically generated - may contain errors
4 protocols using advancestem stem cell growth supplement
1
Adipogenic Differentiation of hUC-MSCs
2
Osteogenic Differentiation of MSCs
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Osteogenic differentiation was induced by treating subconfluent MSC cultures at passage 2 with osteogenic induction medium (AdvanceSTEM osteogenic differentiation medium, supplemented with 10% AdvanceSTEM stem cell growth supplement, Thermo Scientific) for 21 days, according to the manufacturer's instructions and previous report [30 (link)]. Experiments were performed in triplicate. The osteogenic potential was examined for extracellular matrix calcification by von Kossa's method using a commercially available kit (IHC World). Cultures were treated with silver nitrate for 60 min at room temperature under ultraviolet light, followed by treatment with sodium thiosulphate for 5 min. The cells were counterstained with nuclear fast red and then photographed using a phase contrast microscope. The ImageJ software was used for the quantification of the mineralized matrix.
3
Osteogenic Differentiation of MSCs
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Osteogenic differentiation was promoted by treating subconfluent MSC cultures at passage 2 with osteogenic induction medium (AdvanceSTEM osteogenic differentiation medium, supplemented with 10% AdvanceSTEM stem cell growth supplement, ThermoScientific) for 21 days per manufacturer's instructions. Experiments were performed in triplicate. The osteogenic potential was examined for extracellular matrix calcification by the von Kossa's method using a commercially available kit (IHC World). Cultures were treated with silver nitrate for 60 min at room temperature under ultraviolet light, followed by treatment with sodium thiosulphate for 5 min. The cells were counterstained with nuclear fast red and then photographed using phase contrast microscopy. ImageJ software (http://rsbweb.nih.gov/ij/ ) was used for quantification of mineralized matrix.
4
Adipogenic Differentiation of Mesenchymal Stem Cells
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Passage 2 MSCs were seeded in six-well plates in triplicate at a final cell density of 5,000 cells per cm2 and propagated in complete medium. Forty-eight hours later, designated as day 0, differentiation was initiated using adipogenic induction medium (AdvanceSTEM adipogenic differentiation medium supplemented with 10% AdvanceSTEM stem cell growth supplement, ThermoScientific, Rockford, IL), as per the manufacturer's instructions. The medium was changed every 3–4 days thereafter, and experiments were terminated after 3 weeks. The differentiated MSCs were fixed with 4% paraformaldehyde (PFA) and stained with oil red O (IHC World, Woodstock, MD) to visualize accumulated cytoplasmic lipid rich vacuoles.24 (link) The lipoid bodies were observed under phase contrast microscopy in at least 10 nonoverlapping fields. To quantify staining, oil red O was extracted with isopropanol containing 4% nonidet P-40 detergent and optical density was then measured at 490 nm.24 (link)
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