Osteogenesis, adipogenesis, and chondrogenesis were examined to determine the iMSC multipotent differentiation potential. For osteogenic induction, 5 × 104 iMSCs were seeded in 24-well plates until 90% confluence and replaced with osteogenesis medium (Gibco). The cells cultured in MSC medium served as control. After 21 days, the cells were fixed with 4% paraformaldehyde and stained with Alizarin Red to detect areas of mineralized calcium. For adipogenic induction, 5 × 104 iMSCs were seeded in 24-well plates until complete confluence, and differentiation was induced by adipogenesis medium (Gibco). The cells cultured in MSC medium served as the control. After 21 days, the cells were fixed with 4% paraformaldehyde and stained with Oil Red O. For chondrogenic induction, 1 × 106 cells were centrifuged in a 15-mL polypropylene falcon tube to obtain a pellet, and chondrogenic medium (Gibco) was gently added to the pellet. After 21 days, the pellet was fixed with 4% paraformaldehyde and embedded in optimum cutting temperature compound (OCT) (Thermo Fisher, Waltham, MA, USA). Cryosections were stained with Toluidine Blue to detect the presence of proteoglycans. All stained cells were observed under an optical microscope (Leica, Solms, Germany).
Chondrogenic medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Chondrogenic medium is a specialized cell culture medium designed to support the growth and differentiation of chondrocytes, which are the cells responsible for the production of cartilage. The medium provides the necessary nutrients and growth factors to promote the formation of cartilage-like tissue in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Alcian blue, by Merck Group (3 mentions)
Osteogenesis medium, by Thermo Fisher Scientific (2 mentions)
Adipogenesis medium, by Thermo Fisher Scientific (2 mentions)
Optical microscope, by Leica (2 mentions)
Oil red o, by Merck Group (2 mentions)
Alizarin red s, by Merck Group (2 mentions)
Adipogenic medium, by Thermo Fisher Scientific (2 mentions)
Alizarin red solution, by Merck Group (1 mentions)
Oil red o solution, by Merck Group (1 mentions)
Osteogenic induction medium, by Thermo Fisher Scientific (1 mentions)
Flow cytometry analyzer, by Beckman Coulter (1 mentions)
Cm1950, by Leica (1 mentions)
Adipogenic differentiation medium, by Thermo Fisher Scientific (1 mentions)
10 protocols using chondrogenic medium
1
Multilineage Differentiation of iMSCs
2
Multilineage Differentiation of MSCs
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For multilineage differentiation, MSCs at the fourth passage were harvested and replated at a density of 1 × 104 cells/well in a 24-well culture plate. When the cells reached 50 ~ 70% confluency, adipogenic and osteogenic media (Gibco, Billings, MT, USA) were replaced to induce adipogenesis and osteogenesis, respectively. After 21 days, cells were fixed in 4% formaldehyde and stained with Oil Red O (Sigma-Aldrich, St, Louis, MO, USA) or Alizarin Red S (Sigma-Aldrich, St, Louis, MO, USA) to evaluate the adipogenic or osteogenic differentiations, respectively. For chondrogenic differentiation, 2 × 105 WJ-MSCs at the fourth passage were centrifuged for 5 min at 1200 rpm in a tube. The supernatant was removed, and the pellet resuspended in chondrogenic medium (Gibco, Billings, MT, USA). After 21 days, the pellet was fixed in 4% formaldehyde, dehydrated through serial ethanol concentrations, and embedded in optimal cutting temperature compound. Blocks were cut into 5-mm-thick sections and stained with Alcian Blue (Sigma-Aldrich, St, Louis, MO, USA).
3
Chondrogenic Differentiation of NPSCs and BMSCs
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NPSCs and BMSCs were seeded into 6-well plates at a density of 1 × 106 cells/well and induced by adding chondrogenic medium (Gibco, USA). After the medium was changed per the set time points, the cells were collected on the 14th and 21st days. The expression levels of the cartilage marker molecules ACAN and COL2A1 were assessed by RT‒qPCR and Western blot.
4
Chondrogenic Differentiation of C3H/10T1/2 Cells
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C3H/10T1/2 cells were incubated at 2×105 cells/plate and allowed to adhere overnight. The cells were cultured in DMEM containing 2% FBS in the presence of 0.1 mM CoCl2 for 24 or 48 h. Cells were harvested by centrifugation in a 15 ml polypropylene tube, and the pellets were cultured in a chondrogenic medium (Gibco BRL) for 3, 7, 10, and 14 days. The chondrogenic medium was replaced every 3 days. Alcian blue staining was used to detect chondrocyte nodule formation after 14 days of culture. Cells in micromass culture were rinsed with PBS and fixed in 10% formaldehyde in PBS for 20 min. Cultures were washed with water three times and stained in 1% Alcian blue (Sigma-Aldrich) with 3% glacial acetic acid for 24 h. Cultures were de-stained in 70% ethanol two times and stored in water for image capture.
5
Chondrogenic Differentiation of USCs
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Chondrogenic differentiation of USCs was performed using a pellet culture technique. Briefly, 1 × 106 cells were pelleted by centrifugation in a 15-ml centrifuge tube. Chondrogenic medium (Gibco, USA) was gently added to the pellet. After 4 weeks of induction, the pellets were fixed in 4% PFA and embedded in an optimum cutting temperature (OCT) compound. The 10-μm cryosections were stained with Toluidine blue. All of the stained cells were observed under an optical microscope (Leica, Germany).
6
Multilineage Differentiation Assay
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For adipogenic differentiation, the cells were incubated in an adipogenic medium (Gibco, Life Technologies, USA) for two weeks. The cells were then stained with Oil Red O solution (Sigma-Aldrich, USA) for 5 min. For osteogenic differentiation, the cells were incubated in a DMEM–low glucose medium containing 1% A–A, 10-nM dexamethasone, 10-mM β-glycerophosphate, and 50-μM ascorbic acid. After four weeks, the cells were stained with 2% (w/v) Alizarin Red solution (Sigma-Aldrich, USA) for 30 min. Next, the cells were seeded at 1 × 106 cells/mold (StemFIT3D; MicroFIT, Seongnam, Korea) in a normal culture medium to generate a spheroid form for chondrogenic differentiation. After 24 h, the cells in the spheroid form were transferred to a 100-mm cell culture dish and incubated in a chondrogenic medium (Gibco, Life Technologies, USA) for 2 weeks. The medium was changed every 3 d. Then, the cells were stained using Alcian Blue (Sigma-Aldrich, USA) for 30 min while maintaining the spheroid form.
7
Multilineage Differentiation and Characterization of Umbilical Stem Cells
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After the isolation of USCs, we used the Alizarin Red Staining, Oil Red O staining, and Alcian blue staining to verify their multilineage differentiation potential. For osteogenic differentiation, USCs were incubated with an osteogenic induction medium (Gibco). On day 21, after the induction, the cells were stained with Alizarin Red S (Sigma, USA) for observation. For adipogenic differentiation, USCs were incubated with an adipogenic medium (Gibco). On day 21, after the induction, USCs were stained with Oil Red O (Sigma) for observation. For the chondrogenic differentiation, USCs were incubated with a chondrogenic medium (Gibco). After 21 days of induction, allicin blue staining (Sigma) was performed for observation. We also performed the flow cytometry analysis to detect the stem-cell surface markers (CD44, CD73, CD90, CD105, CD31, CD34, and CD45). The USCs were incubated with these antibodies (BD, USA), washed with PBS, and analyzed with a flow cytometry analyzer (Beckman, USA).
8
Tri-lineage Differentiation Evaluation of iMSCs
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Tri-lineage differentiation capability of iMSC was examined as previously described [16 ]. Briefly, to detect osteogenesis, iMSC culture medium was switched to osteogenesis medium (Gibco) when 90% confluency was reached. After culture for 21 days, cells were fixed with 4% (w/v) paraformaldehyde (PFA) and Alizarin Red staining was used to detect mineralized calcium. To detect adipogenesis, iMSC were cultured under adipogenesis medium (Gibco) for 21 days, followed by Oil Red O staining. To detect chondrogenesis, 1 × 106 cells were pelleted in a 15-mL polypropylene tube after centrifugation, and chondrogenic medium (Gibco) was gently added to the pellet. After 28 days, the pellet was fixed with 4% (w/v) PFA and embedded in optimum cutting temperature compound (OCT) (Thermo Fisher, Waltham, MA, USA). Cryosections (8 μm) were cut with freezing microtome (Leica, CM1950, Germany) and stained with Toluidine Blue to examine the presence of proteoglycans. The cells cultured in MSC culture medium were served as control. All images were captured under an optical microscope (Leica, DM6B, Germany).
9
Chondrogenesis of WJ-MSCs in Collagen Scaffold
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Groups of WJ-MSCs in collagen scaffolds were incubated in chondrogenic medium (Gibco, Stempro, Grand Island, NY, USA) with or without different concentrations of the PPARδ agonist, GW0742, or TGF-β for 14 days. The medium was changed every 3 days. Normal rabbit cartilage was used as a control to compare the chondrogenesis of WJ-MSCs in the collagen-based scaffold. The scaffold and rabbit cartilage were fixed with 10% formalin solution and stained with 1% alcian blue to analyze chondrogenesis.
10
Multilineage Differentiation of AMSCs
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Upon reaching 80% confluence, AMSCs at passage 3 were induced and cultured with adipogenic differentiation medium (Gibco). After 14 days, AMSCs were stained with Oil Red O staining and observed under an inverted phase-contrast microscope to assess adipogenic differentiation. In terms of osteogenic differentiation evaluation, the cells were cultured in an osteogenic medium (Gibco) for 21 days followed by Alizarin Red S staining to detect the calcification deposits. For chondrogenic differentiation, 1 × 10 6 cells were centrifuged in a 15 mL centrifuge tube and then transferred to a chondrogenic medium (Gibco). After 4 weeks, the cells were fixed with 4% paraformaldehyde and cut into 10 µm sections. The sections were stained with Toluidine blue and observed under a microscope.
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