Fibroblasts were cultured in adipogenic differentiation medium (Lonza, Basel, Switzerland) for 2 weeks and stained with 1:1,000 HCS LipidTOX™ Deep Red Neutral Lipid Stain (Thermo Fisher Scientific, Waltham, Massachusetts). Images were taken using a Scan R high content screening station (Olympus, Tokyo, Japan). The percentage of differentiated fibroblasts was quantified using Scan R analysis software version 3.1.1. Fibroblasts were cultured in chondrogenic differentiation medium (Promocell, Heidelberg, Germany) for 2 to 3 weeks and time in days was measured until three-dimensional cartilage-like structure formed (Supplementary Figure S7 ).
Adipogenic differentiation medium
Manufactured by Lonza
Sourced in Switzerland
Adipogenic differentiation medium is a laboratory product used to induce and maintain the differentiation of cells, such as mesenchymal stem cells, into adipocytes (fat cells). The medium contains a specific combination of growth factors, hormones, and other components that support the adipogenic differentiation process.
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Lab products found in correlation
Osteogenic differentiation medium, by Lonza (2 mentions)
Chondrogenic differentiation medium, by PromoCell (1 mentions)
Scanr high content screening station, by Olympus (1 mentions)
Hcs lipidtox deep red neutral lipid stain, by Thermo Fisher Scientific (1 mentions)
Formalin, by Merck Group (1 mentions)
Chondrogenic differentiation medium, by Lonza (1 mentions)
5 protocols using adipogenic differentiation medium
1
Adipogenic and Chondrogenic Differentiation
2
Adipogenic Differentiation of AdMSCs
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To differentiate AdMSCs into adipocytes, cells at passage 5-10 were induced in adipogenic differentiation medium (Lonza Group, Ltd.) for 3 weeks. The media were changed every 3-4 days. Melatonin and/or luzindole were added to the tested cells whenever the media were replaced. The differentiated cells were washed with PBS, then incubated with 10% formalin (Sigma-Aldrich; Merck KGaA) for 30 min at room temperature. After washing, 60% isopropanol was dispensed. Oil Red O solution in distilled water was added to the cells for 10 min. After washing with tap water, images of the red-stained cells were obtained using an inverted light phase-contrast microscope (IX71; Olympus Corporation). To obtain quantitative data regarding adipogenesis, absorbance was measured at 500 nm after destaining with isopropanol. Data were expressed as the mean ± SD of three independent experiments.
3
Adipogenic and Osteogenic Potential of MSCs
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The potential of AD- and BM-MSCs to differentiate into adipogenic and osteogenic lineages were examined. To induce adipogenic differentiation, cells were treated with an adipogenic differentiation medium (Lonza, Basel, Switzerland) for 3 weeks. Adipogenesis was assessed by Oil Red O staining (Supplementary Figure S1B ). For osteogenic differentiation, cells were treated with osteogenic differentiation medium (Lonza, Basel, Switzerland) for 3 weeks. Osteogenesis was assessed by Alizarin Red S staining (Supplementary Figure S1C ). Medium changes were performed twice weekly for the two assays.
4
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.
5
Adipogenic Differentiation of Bone Marrow Cells
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Adherent cells derived from bone marrow were cultured in supplemented DMEM-LG medium until confluent. 3 days later, medium was replaced with Lonza Adipogenic differentiation medium, following the manufacturer’s instructions (rMSC Differentiation BulletKit®, Lonza). After 3 weeks, cultures were stained with Oil Red to verify the presence of lipidic vacuoles [18 (link)].
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