The cells were grown to 80% confluence in a 6-well plate and incubated with adipogenic medium (Gibco, USA) that was changed every 3 days. After 21 days of induction, the cells were stained with Oil Red O (Sigma, USA) to visualize lipid vacuoles.
Adipogenic medium
Manufactured by Thermo Fisher Scientific
Sourced in United States
Adipogenic medium is a specialized cell culture medium used to induce and support the differentiation of cells into adipocytes (fat cells). It provides the necessary nutrients and signaling factors to promote the accumulation of lipid droplets within the cells, a hallmark of adipogenic differentiation.
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Lab products found in correlation
Oil red o, by Merck Group (3 mentions)
Chondrogenic medium, by Thermo Fisher Scientific (2 mentions)
Alcian blue, by Merck Group (1 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)
Alizarin red s, by Merck Group (1 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Osteogenic medium, by Thermo Fisher Scientific (1 mentions)
Stempro chondrogenesis differentiation kit, by Thermo Fisher Scientific (1 mentions)
Eclipse e200, by Nikon (1 mentions)
6 protocols using adipogenic medium
1
Adipogenic Differentiation of Cells
2
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.
3
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).
4
Multilineage Differentiation of Cells
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At approximately 20 population doublings, clonally expanded cell lines were lifted from monolayer using TrypLE (Gibco) and centrifuged at 500 ×g for 5 min in 50 mL centrifuge tubes. Following TrypLE aspiration, cells were resuspended in respective differentiation media (osteogenic, adipogenic, or chondrogenic) (Fig. 2). For osteogenic differentiation, cells were suspended in commercial osteogenic medium (Gibco) and plated at 5,000 cells/well in 24well plates and cultured for 21 d (Oikonomopoulos et al., 2015) . For adipogenic differentiation, cells were suspended in adipogenic medium (Gibco) and plated at 30,000 cells/well in 24-well plates and cultured for 21 d (Oikonomopoulos et al., 2015) (link). For fibrochondrogenic differentiation, 100,000 cells/well were pelleted into conical bottom 96-well plates and maintained for 14 d in chondrogenic medium (Anderson et al., 2018; Johnstone et al., 1998) (Fig. 2). Medium was replaced every 2-3 d for all three culture types.
5
Adipogenic Differentiation of HUMSCs
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HUMSCs were treated with adipogenic medium (Gibco-Invitrogen) for 21 days and the medium was refreshed every 3 days. The cells were assessed every 3 days. Adipogenesis was evaluated by Oil Red-O staining. Briefly, the cells were fixed with 4% paraformaldehyde and stained with Oil Red-O (Sigma-Aldrich) for 10 min and observed with inverted phase contrast microscope.
6
Multipotent Differentiation of Rabbit MSCs
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The multipotent capacity of rabbit MSCs was proven after in vitro culturing with specific supplements by inducing differentiation into osteogenic, chondrogenic, and adipogenic. To induce osteogenic differentiation, confluent passaged-2 cells were cultured in the osteogenic medium (Invitrogen, Gibco, USA). After 21 days, Alizarin Red staining was used to observe the matrix mineralization. STEMPRO Chondrogenesis Differentiation Kit (Gibco, Invitrogen) was used to induce chondrogenic differentiation of rabbit MSCs in a micromass pellet culture system. Feeding of chondrogenic medium was carried out over a period of 21 days and the pellet was then processed for histology staining using Safranin O/fast green solution. For adipogenesis, adipogenic medium (Invitrogen, Gibco, USA) was used to induce the differentiation in the confluent culture of passaged-2 cells. Fourteen days after culture initiation, the cells were fixed with methanol at room temperature for 10 minutes, rinsed by 60% isopropanol, and stained by using freshly prepared Oil Red O solution in 99% isopropanol for 15 minutes. The images at different magnifications were captured using a camera attached to the light microscope (Nikon Eclipse E200, Nikon, Japan).
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