MSCs were plated in 96-well plates at a density of 15,000/cm2 for 24 h then media were replaced with either osteoblastogenic culture medium consisting of DMEM supplemented with 10% (v/v) foetal bovine serum (FBS), 10 mM β-glycerol phosphate, 100 nM dexamethasone and 50 µg/ml ascorbic acid 2-phosphate, or adipogenic culture medium consisting of DMEM supplemented with 10% FBS, 1 μM dexamethasone, 10 μg/ml insulin, 0.5 mM IBMX, 60 μM indomethacin, 2 μM rosiglitazone and 20 nM IGF-1 (R&D Systems) (all Sigma unless specified). Media were replaced every 3 or 4 days. Seven days of differentiation was sufficient to assess gene expression changes in markers of differentiation. Cells were cultured for 21 days in osteoblastogenic medium to achieve fully mineralised cultures, and for 14 days in adipogenic medium for lipid production.
Indomethacin
Manufactured by R&D Systems
Sourced in Japan
Indomethacin is a small organic molecule that acts as a non-steroidal anti-inflammatory drug (NSAID). It is commonly used in research applications to inhibit the cyclooxygenase (COX) enzymes, which play a role in the production of prostaglandins and other inflammatory mediators.
Automatically generated - may contain errors
Lab products found in correlation
Rosiglitazone, by R&D Systems (1 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by R&D Systems (1 mentions)
Igf 1, by R&D Systems (1 mentions)
Stemxvivo osteogenic adipogenic base media, by R&D Systems (1 mentions)
Stemxvivo human osteogenic supplement, by R&D Systems (1 mentions)
L name, by Merck Group (1 mentions)
Tgf β1 neutralizing antibody, by R&D Systems (1 mentions)
Ifn γ, by Merck Group (1 mentions)
Glutamate, by Thermo Fisher Scientific (1 mentions)
L ascorbic acid phosphate, by Fujifilm (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Insulin, by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
Follistatin, by R&D Systems (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
5 protocols using indomethacin
1
Multipotent Stromal Cell Differentiation
2
Osteogenic and Adipogenic Differentiation of MSCs
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Osteogenesis was chemically induced by culture in StemXVivo Osteogenic/Adipogenic Base Media (R&D Systems), supplemented with StemXVivo Human Osteogenic Supplement (R&D Systems) and 1% P/S. Adipogenesis was chemically induced in high glucose (4.5 g/L) DMEM with 1 µM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 10 µg/mL insulin and 100 µM indomethacin (all reagents from Sigma), supplemented with 10% FBS and 1% P/S as described previously53 . MSCs were grown in osteogenic, adipogenic or control media for three weeks on collagen-I coated PA hydrogels for RT-qPCR assays, with seeding densities of 2000 and 1000 cells/cm2, for primary and immortalised MSCs respectively (seeding rates were adjusted to account for a greater rate of proliferation in the immortalised cells). For cytochemistry assays, cells were seeded on tissue culture plastic (TCP) at densities of 1200 or 650 cells/cm2, for primary and immortalised MSCs respectively.
3
PBMC Proliferation Assay with LPS and BMSCs
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3H-TdR incorporation assay was performed to evaluate the proliferation capacity of PBMCs isolated from Wistar rats in co-culture with or without LPS or BMSCs. In brief, PBMCs were plated into 96-well plates (2 × 106/ml, 200 μl/well) and treated with or without BMSCs isolated from rats, IL-10-neutralizing antibody (cat. no MAB417, 10 μg/ml, R&D Systems), TGF-β1-neutralizing antibody (cat. no. 1D11, 10 μg/ml, R&D Systems), indomethacin (a COX-2 inhibitor, cat. no. 9758, 20 μM, Sigma-Aldrich), l -NAME (N-nitro-l -arginine methyl ester, an iNOS inhibitor, cat. no. M18168, 1 mM, Meryer, China), 1-MT (1-methyl-l -tryptophan, an IDO inhibitor, cat. no. 860646, 500 μM, Sigma-Aldrich), and antibody IFN-γ (cat. no. ab133566, Abcam, USA) followed by the stimulation of LPS (0.5 μg/ml, Difco Laboratory, USA) or not. After 72 h of incubation, 20 μl 3H-TdR (10 μci/ml) was added into each well for 8 h of incubation. PBMCs were harvested onto glass fiber filter paper, and the counts per minute were determined using a Wallac TriLux 1450 MicroBeta microplate scintillation counter (PerkinElmer, Waltham, MA, USA). All conditions were performed in triplicate.
4
Multilineage Differentiation Potential of MSCs
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To assess the multiple differentiation potential, MSCs were cultured until sub-confluency and induced to differentiate into the adipogenic or osteogenic lineages for 7 and 21 days, respectively. After that, the gene expression analysis and staining were performed. The adipogenic medium consisted of α-MEM (Life Technologies), 20% FBS (Life Technologies), 2 mM glutamate (Life Technologies), 100 Units/mL penicillin/streptomycin (Sigma-Aldrich) and 55 µM 2-ME (Life Technologies), 10 mM L-ascorbic acid phosphate (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 10 mg/mL insulin (Sigma-Aldrich), 0.5 mM hydrocortisone (Sigma-Aldrich), 6 mM indomethacin (R&D systems), 50 mM isobutylmethylxanthin (Enzo life science, Tokyo, Japan), as reported [66 (link)]. Osteogenic medium consisted of α-MEM (Life technologies), 20% FBS (Life Technologies), 2 mM glutamate (Life technologies), 100 Units/mL penicillin/streptomycin (Sigma-Aldrich) and 55 µM 2-ME (Life technologies), 10 mM L-ascorbic acid phosphate (FUJIFILM Wako Pure Chemical Corporation), 200 mM β-glycerophosphate (Sigma-Aldrich), as reported [67 ]. Mineralization (calcium deposition) and lipid droplet formation were evaluated, respectively, by staining with alizarin red-S or oil red-O, as reported [68 (link),69 (link)].
5
Modulation of NK Cell IFN-γ Production
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Umbilical cord-derived mesenchymal stem cells were seeded in 24- or 48-well plates and allowed to adhere for 24 h. Subsequently, freshly isolated allogeneic NK cells were added at a MSC:NK-ratio of 1:10 and co-cultured for 16 h. Cell-free supernatants from the NK-MSC co-cultures or MSC cultures without NK cells were frozen to analyze the role of soluble factors as MSC-NK-conditioned medium (NK-MSC cm) or MSC-conditioned media (MSC cm), respectively. NK cells exposed to MSCs or different conditioned media were collected, and tested for IFN-γ production. NK cells cultured alone in unconditioned media were used as controls. To induce IFN-γ production, NK cells were stimulated with IL-12 (10 ng/ml) or IL-18 (10 ng/ml) alone or in combination. Brefeldin A (Sigma) was added after 1 h, and the incubation was continued for 3 additional hours. IFN-γ production was analyzed by intracellular cytometric analysis. To neutralize activin-A, follistatin (R&D Systems) was added to MSC cm at a final concentration of 0.4 μg/ml. To block PGE2 production, indomethacin (R&D Systems) was added to MSC cultures at a concentration of 20 μM. Cell-free supernatants were generated from these cultures and are referred to as INDO-MSC cm. MSC cm was also collected from the same MSCs as controls.
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