Cells were plated in a 6-well plate (Corning Life Sciences) at a density of 50,000 cells/cm2 in culture media and placed in an incubator at 37°C/5% CO¬2. After 24 h, media in control wells was replaced, media in experimental wells was aspirated, and osteocyte differentiation medium (StemPro Osteogenic Differentiation Medium, Gibco) was added. Cells were fed with their corresponding media every 3 days. After a minimum of 2 weeks, media was aspirated from the wells, the cells were washed with DPBS, 1 mL of 10% formalin (ThermoFisher) was added to each well and incubated overnight to fix the cells. The formalin was aspirated and the wells were washed with DI water, the wash fluid was aspirated, and 1 mL of 1% Alizarin Red staining solution (Sigma-Aldrich, St. Louis, MO, USA) was added to each well for 30 min at room temperature with gentle rocking. The stain was aspirated from the wells and the plate was washed with DI water to remove excess stain. After drying at room temperature, the wells were imaged with a phase contrast microscope fitted with an Amscope MD camera.
Stempro osteogenic differentiation medium
Manufactured by Thermo Fisher Scientific
StemPro Osteogenic Differentiation Medium is a cell culture medium designed for the differentiation of stem cells into osteoblasts. It provides the necessary components to support the osteogenic differentiation process.
Automatically generated - may contain errors
Lab products found in correlation
Formalin, by Merck Group (1 mentions)
Alizarin red staining solution, by Merck Group (1 mentions)
6 well plate, by Corning (1 mentions)
Stempro adipogenic differentiation medium, by Thermo Fisher Scientific (1 mentions)
Stempro chondrogenic differentiation medium, by Thermo Fisher Scientific (1 mentions)
2 protocols using stempro osteogenic differentiation medium
1
Alizarin Red Staining for Osteogenesis
2
Mesodermal Differentiation of Adventitial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
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For osteogenic differentiation, CD10 + and CD10 -adventitial cells at 70% confluence were cultivated in STEMPRO osteogenic differentiation medium (Gibco). After 28 days, cells were fixed in 4% PFA for 2 minutes and incubated for 10 minutes with alizarin red (pH 4.2) or von Kossa reagent for detection of calcium deposits.
For chondrogenesis, high-density pellets were prepared by spinning down 5 × 10 5 cultured cells in 15 mL conical tubes and grown in STEMPRO chondrogenic differentiation medium (Gibco) for 21 days.
Pellets were fixed in 10% formalin, dehydrated using a graded series of ethanol washes, and embedded in paraffin. Five-micrometer thick sections were rehydrated and stained with alcian blue and nuclear fast red for detection of sulfated glycosaminoglycans and nuclei, respectively.
For adipogenic differentiation, CD10 + and CD10 -adventitial cells at 70% confluence were cultured in STEMPRO adipogenic differentiation medium (Gibco) for 14 days. Cells were fixed in 2% PFA at RT, washed in 60% isopropanol, and incubated with oil red O for 10 minutes at RT for detection of lipids.
All mesodermal differentiation assays were performed in technical triplicates, and cells cultured in basal medium were used as negative controls.
For chondrogenesis, high-density pellets were prepared by spinning down 5 × 10 5 cultured cells in 15 mL conical tubes and grown in STEMPRO chondrogenic differentiation medium (Gibco) for 21 days.
Pellets were fixed in 10% formalin, dehydrated using a graded series of ethanol washes, and embedded in paraffin. Five-micrometer thick sections were rehydrated and stained with alcian blue and nuclear fast red for detection of sulfated glycosaminoglycans and nuclei, respectively.
For adipogenic differentiation, CD10 + and CD10 -adventitial cells at 70% confluence were cultured in STEMPRO adipogenic differentiation medium (Gibco) for 14 days. Cells were fixed in 2% PFA at RT, washed in 60% isopropanol, and incubated with oil red O for 10 minutes at RT for detection of lipids.
All mesodermal differentiation assays were performed in technical triplicates, and cells cultured in basal medium were used as negative controls.
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