Cultured ACL- and BM-MSCs at passage 3 were harvested using trypsin–EDTA (Gibco), transferred to a 24-well plate and grown overnight in culture medium. The cell numbers used were as follows: 2.0 × 104 cells (for adipogenesis) and 1.5 × 104 cells (for osteogenesis). For adipogenic differentiation, adherent cells were cultured in adipogenic induction and maintenance medium (Lonza), which was changed every 3–4 days. After 14 days, Oil red O staining (Muto Pure Chemicals) was used to confirm the differentiation of the cells into adipocytes. For osteogenic differentiation, adherent cells were cultured in osteogenic induction medium (Lonza), which was changed every 3–4 days. After 14 days, the differentiation of the cells into osteoblasts was assessed by Alizarin Red staining (Millipore). For chondrogenic differentiation, 2.0 × 105 cells were transferred to a 1.5 mL tube and cultured in chondrogenic induction medium (Lonza) containing 10 ng/mL TGF-β3 (Lonza) and 500 ng/mL BMP-6 (R&D Systems), which was changed every 3–4 days.
Oil red o staining
Manufactured by Muto Pure Chemicals
Sourced in Japan
Oil Red O is a fat-soluble dye used for staining neutral lipids and triglycerides in histological samples. It has the chemical formula C26H24N2O.
Automatically generated - may contain errors
Lab products found in correlation
Alizarin red staining, by Merck Group (3 mentions)
Osteogenic induction medium, by Lonza (2 mentions)
Chondrogenic induction medium, by Lonza (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Tgf β3, by Lonza (1 mentions)
Adipogenic induction medium, by Lonza (1 mentions)
Toluidine blue, by Fujifilm (1 mentions)
Bone morphogenetic protein 6, by R&D Systems (1 mentions)
Transforming growth factor β3, by Lonza (1 mentions)
Crystal violet staining, by Merck Group (1 mentions)
Indomethacin, by Fujifilm (1 mentions)
Dexamethasone (dex), by Fujifilm (1 mentions)
Bone morphogenetic protein 2, by Medtronic (1 mentions)
15 ml tube, by BD (1 mentions)
Isobutylmethylxanthine, by Merck Group (1 mentions)
Ascorbic acid 2 phosphate, by Fujifilm (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
Transforming growth factor β3, by Miltenyi Biotec (1 mentions)
4 protocols using oil red o staining
1
Differentiation of Mesenchymal Stem Cells
2
Multilineage Differentiation of Cultured MSCs
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Cultured MSCs at three passages were harvested using Trypsin-EDTA (Gibco). For adipogenic differentiation, 1.0 × 104 cells were transferred to a 24-well plate and cultured overnight in culture medium. Adherent cells were cultured in adipogenic induction medium (Lonza), which was changed every 3–4 days. After 14 days, oil red O staining (Muto Pure Chemicals) confirmed the differentiation of these cells into adipocytes. For osteogenic differentiation, 7.0 × 103 cells were transferred to a 24-well plate and cultured overnight in culture medium. Adherent cells were cultured in osteogenic induction medium (Lonza), which was changed every 3–4 days. After 14 days, the differentiation of these cells into osteoblasts was assessed by alizarin red staining (Millipore). For chondrogenic differentiation, 5.0 × 105 cells were transferred to a 15-mL tube and cultured in chondrogenic induction medium (Lonza) containing 10 ng/mL transforming growth factor-β3 (Lonza) and 500 ng/mL bone morphogenetic protein 6 (R&D Systems), which was changed every 3–4 days. After 21 days, chondrogenic differentiated cells were was analyzed by Toluidine blue (Wako) and Safranin O staining (TCI) staining.
3
Immunofluorescence Staining of Cells
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For immunofluorescence staining, cells were seeded on a chamber slide. After 2 days, they were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 solution, and blocked with 5% BSA. They were incubated with primary antibodies (1 h) and then with secondary antibody (1 h). BODIPY and DAPI were used for cytochemical staining for LDs and nucleoli, respectively. Oil Red O staining (Muto pure chemicals, Japan) and crystal violet staining (Merck) was used for detection of lipid droplet and living cells, respectively.
4
Multilineage Differentiation Potential of Synovial MSCs
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For adipogenesis, synovial MSCs were plated at 100 cells/60-cm2 dish and cultured for 14 days in culture medium. The adherent cells were cultured in adipogenic induction medium supplemented with 100 nM dexamethasone, 0.5 mM isobutyl-methylxanthine (Sigma-Aldrich), and 50 mM indomethacin (Wako) for an additional 21 days. Adipocytes were stained with oil red-o staining (Muto Pure Chemicals, Tokyo, Japan).
For calcification, 100 cells were transferred to a 60-cm2 dish and cultured for 14 days in culture medium. The adherent cells were cultured in calcification induction medium containing 50 μg/mL ascorbic acid 2-phosphate (Wako), 10 nM dexamethasone (Wako), and 10 mM β-glycerophosphate (Sigma-Aldrich). After 21 days, calcification was assessed by alizarin red staining (Merck Millipore, Billerica, MA, USA).
For chondrogenesis, 250,000 synovial MSCs were transferred to a 15-mL tube (BD Falcon) and cultured in chondrogenic induction medium containing 10 ng/mL transforming growth factor-β3 (Miltenyi Biotec K.K., Tokyo, Japan) and 1 μg/mL bone morphogenetic protein 2 (Medtronic, TN, USA), which was changed every 3–4 days. After 21 days, cartilage pellets were weighed and evaluated histologically.
For calcification, 100 cells were transferred to a 60-cm2 dish and cultured for 14 days in culture medium. The adherent cells were cultured in calcification induction medium containing 50 μg/mL ascorbic acid 2-phosphate (Wako), 10 nM dexamethasone (Wako), and 10 mM β-glycerophosphate (Sigma-Aldrich). After 21 days, calcification was assessed by alizarin red staining (Merck Millipore, Billerica, MA, USA).
For chondrogenesis, 250,000 synovial MSCs were transferred to a 15-mL tube (BD Falcon) and cultured in chondrogenic induction medium containing 10 ng/mL transforming growth factor-β3 (Miltenyi Biotec K.K., Tokyo, Japan) and 1 μg/mL bone morphogenetic protein 2 (Medtronic, TN, USA), which was changed every 3–4 days. After 21 days, cartilage pellets were weighed and evaluated histologically.
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