For osteogenic and adipogenic differentiation, SFM-expanded hUC-MSCs at the 10th passage were seeded in 24-well plates at a concentration of 5 × 104 cells per well. The StemPro Adipogenesis Differentiation Kit (A10070-01; GIBCO, Grand Island, NY, USA) and the Osteogenesis Differentiation Kit (A10072-01; GIBCO) were used as the differentiation-inducing medium. The medium was refreshed twice every week. After 21 days of differentiation, cells were fixed in 70% ethanol and stained with Alizarin Red S (for osteogenic differentiation) or Oil Red O (for adipogenic differentiation). For chondrogenic differentiation, 4 × 105 SFM-expanded hUC-MSCs at the 10th passage were suspended in 1 ml StemPro Chondrogenesis Differentiation Kit (A10071-01; GIBCO) and distributed to 15 ml centrifuge tubes. Cells were centrifuged at 500 × g for 5 minutes and then placed in an incubator with the caps loosened. The chondrogenic culture was refreshed twice every week. After 21 days of chondrogenesis, the pellets were fixed in 4% formaldehyde, cut to 5 μm and stained by Toluidine blue.
Osteogenesis differentiation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Osteogenesis Differentiation Kit is a laboratory tool used to induce and study the differentiation of cells into osteoblasts, the cells responsible for bone formation. The kit provides a standardized, optimized medium and supplements to guide the differentiation process.
Automatically generated - may contain errors
Lab products found in correlation
Alizarin red s, by Merck Group (3 mentions)
Chondrogenesis differentiation kit, by Thermo Fisher Scientific (2 mentions)
Adipogenesis differentiation kit, by Thermo Fisher Scientific (2 mentions)
Stempro adipogenesis differentiation kit, by Thermo Fisher Scientific (1 mentions)
Stempro chondrogenesis differentiation kit, by Thermo Fisher Scientific (1 mentions)
6 well culture plate, by Thermo Fisher Scientific (1 mentions)
Oil red o staining, by Merck Group (1 mentions)
Chondrogenic differentiation kit, by Thermo Fisher Scientific (1 mentions)
9 protocols using osteogenesis differentiation kit
1
Multilineage Differentiation of hUC-MSCs
2
Osteogenic Differentiation and Alizarin Red Staining
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When cells reached the proper confluence, the medium was replaced to differentiation medium from an Osteogenesis Differentiation Kit (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). After 21 days of differentiation, cells were fixed with 4% PFA for 30 min and washed with PBS. Fixed cells were washed twice with distilled water and then stained with 2% Alizarin Red S (Sigma-Aldrich, Saint Louis, MO, USA To stain the cells, dye solution was applied for 3 min prior to rinsing with distilled water.
3
Mesenchymal Stem Cell Differentiation
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The cMSCs in complete culture media containing 0.5% FBS were pipetted onto a GRGDSP(FN) coated 100mm culture dish (SPL). When 70% confluency was reached, all trypsinized cells were pipetted onto a 6-well culture plate (Nunc, Roskilde, Denmark) at a concentration of 1×105 cells/well. When 70% confluency was reached in the growth medium, the cells were placed in a differentiation medium and incubated at 37 ℃ for 21d. The differentiation medium was replaced twice weekly. Osteogenic differentiation was performed according to the manufacturer's instructions using an Osteogenesis Differentiation Kit (Gibco), and calcium deposits were subsequently visualized using the Von Kossa Method. Cells were fixed with 4% paraformaldehyde, placed in a 3% silver nitrate solution, and exposed to UV light for 60 min. After rinsing with distilled water 3 times, they were then placed in 5% sodium thiosulfate for 2 min.
Chondrogenic differentiation was performed according to the manufacturer's instructions using a Chondrogenesis Differentiation Kit (Gibco). Chondrogenic differentiation was visualized by measuring metachromatic matrix using Alcian blue staining. Cells were fixed with 4% paraformaldehyde and placed in 1% Alcian blue solution for 30 min at room temperature. After rinsing twice with distilled water, the nuclei were stained using Nuclear Fast Red for 5 min.
Chondrogenic differentiation was performed according to the manufacturer's instructions using a Chondrogenesis Differentiation Kit (Gibco). Chondrogenic differentiation was visualized by measuring metachromatic matrix using Alcian blue staining. Cells were fixed with 4% paraformaldehyde and placed in 1% Alcian blue solution for 30 min at room temperature. After rinsing twice with distilled water, the nuclei were stained using Nuclear Fast Red for 5 min.
4
Osteogenic Differentiation of hASCs
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We used a commercially available osteogenesis differentiation kit (Gibco) as directed by the manufacturer to induce hASC osteogenesis. After incubating in differentiation medium for 3 weeks, the cells were fixed in 4% paraformaldehyde for 1 h at room temperature and washed at least 3 times with distilled water. The cells were then stained with 1 ml of von Kossa reagent (Leagene, Beijing, China) under UV light for 10 min.
5
Multilineage Differentiation of HUCMSCs
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Cells were cultured in osteogenic, adipogenic, or chondrogenic differentiation medium, respectively [osteogenesis differentiation kit, adipogenic differentiation kit, chondrogenic differentiation kit (Gibco, Carlsbad, CA, USA)], in 6-well plates for 2–3 weeks for HUCMSC differentiation. During this time, the medium was changed every 2–3 days. Cells were fixed with 4% formaldehyde for 30 min and stained with Alizarin Red S solution (pH 4.2) for 10 min. Osteogenic differentiation was observed under a microscopy (Nikon, Tokyo, Japan). Adipogenic differentiation was dyed using Oil Red O staining (Sigma-Aldrich, St Louis, MO, USA). Cartilage differentiation was determined using Alcian Blue staining.
6
Osteogenic Differentiation and Alizarin Red Staining
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After the cells reached a proper confluence, the standard culture medium was changed to differentiation medium from an Osteogenesis Differentiation Kit (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). After 21 days of differentiation, cells were fixed with 4% PFA for 30 min and then washed with PBS. The cells were washed twice with distilled water and stained with 2% Alizarin Red S (Sigma-Aldrich, Saint Louis, MO, USA). In order to stain the cells, dye solution was applied for 3 min before rinsing with distilled water.
7
Multilineage Differentiation of UC-MSCs
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Adipogenic and osteogenic differentiation assays at the fourth passage were conducted to detect the multilineage differentiation potential of the isolated human UC‐MSCs. UC‐MSCs were induced to differentiate using an Adipogenesis Differentiation Kit (A1007001, Gibco) and Osteogenesis Differentiation Kit (A1007201, Gibco). Lipid accumulation was detected at 7–14 days after induction culture by Oil Red O staining. For osteogenic differentiation, the calcium deposition was detected at 21–28 days after induction culture by Alizarin Red S staining.
8
Osteogenic Differentiation of MSCs
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To induce differentiation into osteogenic lineage, the Osteogenesis Differentiation Kit (Gibco) was used. After 21–25 days of culture in specific medium replaced every 3 days, WJ-MSC and AD-MSC were fixed with 4% PFA for 30 minutes.
Fixed cells were washed twice with distilled water and then stained with 2% Alizarin Red S (Sigma-Aldrich). For staining of the cultures, prepared dye solution was applied for 2-3 minutes and cells were rinsed with distilled water at the end.
Fixed cells were washed twice with distilled water and then stained with 2% Alizarin Red S (Sigma-Aldrich). For staining of the cultures, prepared dye solution was applied for 2-3 minutes and cells were rinsed with distilled water at the end.
9
Multilineage Differentiation of Bcl-2-ADSCs
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To induce adipogenic differentiation, Bcl-2-ADSCs were seeded onto plates at 1 × 10 4 cells/cm 2 in complete DMEM. After culturing for 24 h, the medium was changed to adipogenic induction medium using an Adipogenesis Differentiation Kit (Gibco) and the cells were cultured for 14 days. Then, Oil Red O staining was used to observe oil droplets in the cytoplasm of cells. For chondrogenic differentiation, 5-μL droplets of Bcl-2-ADSCs in complete DMEM (1 × 10 7 cells/mL) were seeded onto plates. After culturing for 48 h, the medium was changed to chondrogenic induction medium using a Chondrogenesis Differentiation Kit (Gibco). Chondrogenic differentiation was induced for 14 days. Chondrogenic differentiation was assessed using Alcian Blue staining. For osteogenic differentiation, Bcl-2-ADSCs (5 × 10 3 /cm 2 ) were seeded onto plates in complete DMEM. After culturing for 48 h, the medium was changed to osteogenic induction medium using an Osteogenesis Differentiation Kit (Gibco) and cells were cultured for 24 days. The cells were then stained with a 1% Alizarin Red S solution.
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