To promote osteogenic, chondrogenic, and adipogenic differentiation, hPDLSCs were cultured in StemPro Osteogenic, StemPro Chondrogenic, and StemPro Adipogenic differentiation medium (Gibco BRL), respectively, with the appropriate supplements as previously reported [17 (link)]. At 21 days, the cells with post-osteogenic, post-chondrogenic, and post-adipogenic induction were stained with 2% Alizarin Red S stain at pH 4.2 (Sigma-Aldrich), 1% Alcian Blue (Sigma-Aldrich), and 0.3% Oil Red O dye (Sigma-Aldrich) to detect proteoglycans, Nissl bodies, and fat vacuoles as indicators of osteogenic, chondrogenic, and adipogenic differentiation, respectively. Stained cells were observed and those were visualized under an inverted light microscope (Olympus U-SPT; Olympus).
Stempro adipogenic differentiation medium
Manufactured by Thermo Fisher Scientific
StemPro® adipogenic differentiation medium is a culture medium designed to support the adipogenic differentiation of stem cells. It provides the necessary components to facilitate the conversion of stem cells into adipocytes (fat cells).
Automatically generated - may contain errors
Lab products found in correlation
L ascorbic acid 2 phosphate, by Merck Group (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
β glycerophosphate, by Merck Group (2 mentions)
Oil red o dye, by Merck Group (1 mentions)
Alcian blue, by Merck Group (1 mentions)
Alizarin red s stain, by Merck Group (1 mentions)
U spt, by Olympus (1 mentions)
Stempro chondrogenic, by Thermo Fisher Scientific (1 mentions)
Stempro osteogenic differentiation medium, by Thermo Fisher Scientific (1 mentions)
Stempro chondrogenic differentiation medium, by Thermo Fisher Scientific (1 mentions)
Oil red o staining, by Merck Group (1 mentions)
Cetylpyridinium chloride, by Merck Group (1 mentions)
Alizarin red s staining, by Merck Group (1 mentions)
Oil red o, by Merck Group (1 mentions)
Isopropanol, by Merck Group (1 mentions)
5 protocols using stempro adipogenic differentiation medium
1
Multilineage Differentiation of hPDLSCs
2
Mesodermal Differentiation of Adventitial Cells
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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.
3
Adipogenic Differentiation of GPCs
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The ability of GPCs to differentiate into multiple stromal lineages was tested as previously described (Mohamed-Ahmed et al., 2018 (link)). Briefly, for adipogenic differentiation, cells in HPL and FBS were cultured in StemPro® adipogenic differentiation medium (Invitrogen) or standard growth medium (control). After 21 days, cells were fixed with 4% paraformaldehyde (PFA) for 10 min at RT and intracellular lipid formation was assessed via Oil red O staining (Sigma-Aldrich, St. Louis, MO, United States).
4
Stromal Lineage Differentiation Assay
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The ability of BMSCs and ASCs to differentiate into multiple stromal lineages was tested as previously described [17 (link)]. Briefly, for adipogenic differentiation, cells in HPL and FBS were cultured in StemPro® adipogenic differentiation medium (Invitrogen) or standard growth medium (control). After 14 days, intracellular lipid formation was assessed via Oil red O (Sigma-Aldrich) staining. For quantification, the stain was extracted using 99% isopropanol (Sigma-Aldrich) and absorbance was measured at 540 nm using a microplate reader. For osteogenic differentiation, cells in HPL and FBS were cultured in osteogenic differentiation medium prepared by adding final concentrations of 0.05 mM L-ascorbic acid 2-phosphate, 10 nM dexamethasone, and 10 mM β glycerophosphate (all from Sigma-Aldrich) to the respective growth media. Cells in standard growth medium served as controls. After 21 days, extracellular calcium deposition was evaluated via Alizarin red S staining (Sigma-Aldrich). For quantification, the stain was dissolved in cetylpyridinium chloride (Sigma-Aldrich) and absorbance was measured at 540 nm using the microplate reader.
5
Mesenchymal Stem Cell Differentiation
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For osteogenic differentiation, ASCs and BMSCs were seeded in 12-well plates at a density of 3 × 103 cells/cm2. After 24 h, ASCs and BMSCs were washed with PBS and osteogenic differentiation medium was added. Osteogenic differentiation medium was prepared by adding 0.05 mM L-ascorbic acid 2-phosphate, 10 nM dexamethasone and 10 mM β glycerophosphate (all from Sigma-Aldrich) to the culture medium. ASCs and BMSCs in routine culture medium served as control. For chondrogenic differentiation, ASCs and BMSCs were seeded in 15 ml tubes at a density of 5 × 105 cells to form a pellet. After 24 h, pellets were washed with PBS and StemPro® chondrogenic differentiation medium (Invitrogen) was added. BMSCs and ASCs pellets in routine culture medium served as control. All media were changed twice per week for 4 weeks. For adipogenic differentiation, ASCs and BMSCs were seeded in 12-well plates at a density of 7 × 103 cells/cm2. After 24 h, ASCs and BMSCs were washed with PBS and StemPro® adipogenic differentiation medium (Invitrogen) was added. ASCs and BMSCs in routine culture medium served as control. Adipogenic and control media were changed twice per week for 2 weeks.
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