Bone marrow stem cells were labeled with Ferumoxytol or MegaPro nanoparticles using the method above and seeded in twelve well plates and cultured in chondrogenic differentiation medium (Lonza, Hayward, CA) at 10×106 cells/ml for 21 days. The cells were replenished with fresh chondrogenic medium every other day. At day 21, the medium was removed and the micromass were fixed in 4% paraformaldehyde before staining with Alcian blue solution (Sigma, St. Louis, MO). The accumulation of proteoglycans in these cells were visualized by imaging (Keyence, BZ-X710, Cupertino, CA). Please also refer to Figure 1 I-K.
Chondrogenic differentiation medium
Manufactured by Lonza
Sourced in Switzerland
Chondrogenic differentiation medium is a cell culture medium designed to support the differentiation of cells, specifically the chondrogenic lineage. The core function of this medium is to provide the necessary nutrients and growth factors to promote the development of chondrocytes, the cells responsible for the formation of cartilage tissue.
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Lab products found in correlation
Ascorbic acid 2 phosphate, by Merck Group (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Indomethacin, by Merck Group (2 mentions)
β glycerophosphate, by Merck Group (2 mentions)
Ascorbic acid 3 phosphate, by Merck Group (2 mentions)
Bz x710, by Keyence (1 mentions)
Alcian blue solution, by Merck Group (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Iq5 real time pcr detection system, by Bio-Rad (1 mentions)
Interleukin 1β il 1β, by Thermo Fisher Scientific (1 mentions)
Moloney murine leukemia virus reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Adipogenic differentiation medium, by Lonza (1 mentions)
Osteogenic differentiation medium, by Lonza (1 mentions)
Dmem low glucose, by Corning (1 mentions)
Alcian blue, by Merck Group (1 mentions)
Oil red o, by Merck Group (1 mentions)
Alkaline phosphatase staining, by Beyotime (1 mentions)
8 protocols using chondrogenic differentiation medium
1
Chondrogenic Differentiation of Bone Marrow Stem Cells
2
Redifferentiation and Transduction of Primary Chondrocytes
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Primary normal human articular chondrocytes (Lonza) were redifferentiated in alginate beads per the manufacturer's protocol using chondrogenic differentiation medium (Lonza). Redifferentiated normal human articular chondrocytes were transduced with lentiviruses expressing GFP or PEDF for 48 h, then cultured for 4 days in chondrogenic differentiation medium in the presence or absence of 1 ng/mL of interleukin-1β (IL-1β) (PeproTech). Total RNA from cultured chondrocytes was isolated using the RNeasy Mini Kit (Qiagen) and complementary DNA was generated using Moloney Murine Leukemia Virus Reverse Transcriptase (Invitrogen). Quantitative PCR was performed using an iQ5 Real-Time PCR Detection System (Bio-Rad). All RT-PCR analyses were normalized to TATA box binding protein mRNA expression [33 (link)]. Primer sequences are available from the author upon request.
3
Multilineage Differentiation of hAFSCs
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To investigate the differentiation ability, hAFSCs were differentiated in vitro into osteogenic, adipogenic, and chondrogenic lineages. hAFSCs were independently cultured either in “adipogenic differentiation medium,” “osteogenic differentiation medium,” or “chondrogenic differentiation medium” (Lonza, Basel, Switzerland) at 37°C in 5% CO2 for the appropriate time according to the manufacturer's recommended protocol. Osteogenesis was assessed by Alizarin staining (Cosmo Bio Co., Ltd., Tokyo, Japan) of the calcified extracellular matrix deposition. Oil Red O staining was used to detect intracellular lipid droplet formation to evaluate adipogenesis. Chondrogenic differentiation was determined by Alcian blue staining.
4
Characterization of hUCB-MSCs Differentiation
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hUCB-MSCs were characterized by the cell surface protein profile and the differentiation capability. The harvested cells were analyzed for the expression of CD73, CD105, CD29, CD44, HLA-ABC, CD90, CD13, CD166, CD45, CD34, HLA-DR by flow cytometry with FACS laser cytometer (Beckman Coulter, Seoul, Korea). Approximately 0.5–1 × 105 cells per tube were used for cell surface antigen expression studies. To evaluate differentiation capability of hUCB-MSCs, multi lineage differentiation tests were performed in vitro. For induction of osteogenic or adipogenic differentiation, 70% confluent cells were incubated in respective differentiation medium (Lonza). For induction of chondrogenic differentiation, the hUCB-MSCs pellet was incubated in a polypropylene tube containing commercially available chondrogenic differentiation medium (Lonza).
After 3 weeks, the osteogenic differentiation ability was assessed by Von Kossa staining. After 4 weeks, adipogenic differentiation was assessed by oil red O staining. After 6 weeks, the harvested chondrogenic pellets were fixated with 10% formalin and paraffin embedded for histological processing. 5-μL-thin sections were stained with safranin O.
After 3 weeks, the osteogenic differentiation ability was assessed by Von Kossa staining. After 4 weeks, adipogenic differentiation was assessed by oil red O staining. After 6 weeks, the harvested chondrogenic pellets were fixated with 10% formalin and paraffin embedded for histological processing. 5-μL-thin sections were stained with safranin O.
5
Multilineage Differentiation Assay
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Adherent cells at P4 were induced toward adipogenic, osteogenic and chondrogenic differentiation, as previously described (Cicione et al., 2016 (link)). Adipogenic medium was prepared by adding to DMEM-low glucose the following: 10% FBS, 1 µM dexamethasone, 0.5 mM 3–isobutyl-1-methylxanthine (IBMX), 10 ug/mL insulin and 100 µM indomethacin. On the other hand, osteogenic medium composition was prepared by adding to DMEM-low glucose (Corning) the following: 10% FBS, 0.1 µM dexamethasone, 0.2 mM ascorbic acid 2-phosphate, and 10 mM glycerol 2-phosphate. With regards to chondrogenic differentiation, cells were digested with trypsin, washed with high glucose-DMEM and were resuspended in Chondrogenic Differentiation Medium (Lonza, Basel, Switzerland) after centrifugation at 300 g for 5 min. After 21 days, cell differentiation was assessed morphologically using Oil Red-O staining for cytoplasmic lipid droplets, Alizarin red staining for mineralized matrix and Alcian blue staining for cartilaginous ECM. The pellets were fixed in 10% neutral buffered formalin and embedded in paraffin and observed after staining as described above.
6
Adipogenic and Chondrogenic Differentiation of 2H11 Cells
7
Multilineage Differentiation Assay
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Cells were cultured in twelve-well plates at a density of 1 × 105 cells per well. To induce adipogenic differentiation, the medium was replaced with basal α-modification of Eagle's medium plus 100 nM dexamethasone, 50 μg/mL ascorbic acid 3-phosphate, and 50 μg/mL indomethacin (Sigma-Aldrich, St. Louis, MO, USA) for 2 weeks. The differentiated cells were fixed with 4% polyoxymethylene for 15 min before staining with 0.3% Oil Red O (Sigma-Aldrich) solution to evaluate adipogenesis. To induce chondrogenic differentiation, the cells were incubated in the presence of chondrogenic differentiation medium (Lonza, Basel, Switzerland) with recombinant transforming growth factor beta-3 protein (R&D Systems, Minneapolis, MN, USA) for 2 weeks. The induced cells were fixed with 4% polyoxymethylene for 15 min, followed by staining with 1% Alcian blue (Sigma-Aldrich). To induce osteogenic differentiation, the cells were incubated in the presence of 10 nM dexamethasone, 50 mg/mL ascorbic acid 2-phosphate, and 10 mM β-glycerophosphate (all from Sigma-Aldrich) for 5 days. Then, the differentiated cells were fixed with 4% polyoxymethylene for 15 min, followed by alkaline phosphatase staining (Beyotime) to assess mineral deposition according to the manufacturer's instructions.
8
Multilineage Differentiation of Stem Cells
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Cells were planked into plates containing basal complete medium and cultured to 80%–90% confluency. The basal medium was then replaced with the corresponding differentiation-inducing mixture. The medium was replaced with a DMEM with low glucose (Hyclone, New York, United States) complete medium containing 10 nM dexamethasone (Sigma, United States), 50 mg/mL ascorbic acid 2-phosphate (Sigma, United States), and 10 mM β-glycerophosphate (Sigma, United States) to induce osteogenic differentiation. Adipogenic differentiation induction medium contained 100 nM dexamethasone (Sigma, United States), 50 μg/mL ascorbic acid 3-phosphate (Sigma, United States), and 50 μg/mL indomethacin (Sigma, United States). Chondrogenic differentiation was induced using a chondrogenic differentiation medium (Lonza, Basel, Switzerland) supplemented with recombinant TGFb3 protein (R&D Systems, Minneapolis, MN, United States) (Li et al., 2020 (link)).
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