Efficient differentiation of intermediate lineages into vascular SMCs.
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The SMC subtypes, namely the neuroectoderm-derived SMC, lateral mesoderm-derived SMC, paraxial mesoderm-derived SMC are abbreviated as NE-SMC, LM-SMC and PM-SMC respectively.
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ELISA of PDGF-BB secreted by EPCs in response to PDGFR-β transfection.
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The concentration of PDGF-BB in the supernatant of culture medium was measured using ELISA.
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Effect of calcitriol stimulation on VDR mRNA transcript, VDR protein expression, cell proliferation in PCASMCs.
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Quiescent PCASMCs were stimulated with 20 ng/ml PDGF-BB with calcitriol for 24 h, and BrdU incorporation was analyzed .
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Growth factor treatment and cell proliferation assay
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Undifferentiated MVSCs and partially differentiated MVSCs (cultured in DMEM with 10% FBS for 3 weeks) were starved in DMEM with 1% FBS for 24 hours followed by the treatment of 10 ng/ml bFGF , 10 ng/ml PDGF-B or 10 ng/ml TGF-β1 for another 24 hours.
- The cell proliferation was quantified by using Click-iT EdU Alexa Fluor 488 HCS Assay kit according to the instruction of the manufacturer.
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Primary Culture of Epicardial Cells and Cardiomyocytes
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Primary epicardial cells were cultured in 10% FBS/MEM containing recombinant soluble factors, such as TGFβ1 (10 ng/ml&systems), FGF2 (100 ng/mloche), , , , , PGF-BB (100 ng/ml) or retinoic acid (1 µmol/l), for 3 days.
- The soluble factors were added on the day of heart removal.
- The medium was replaced daily.
- For western blot analysis, cells were treated with TGFβ1 for 2 days, beginning on the day following heart removal, at a final concentration of 1 ng/ml.
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In vitro culture of stromal cells
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Stromal cells were derived from E14.5 p190-B−/− and WT fetal livers.
- The cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM) containing 20% fetal bovine serum (FBS, Omega Scientific, Tarzana, CA) and beta mercaptoethanol (2-ME , , ).
- The adherent cells were grown to 70-80% confluence, passaged and sub-cultured in medium containing EGF (10ng/ml ) and PDGF (20ng/ml) .
- The cells were depleted for CD11b using lineage depletion kit as per the manufacturer's protocol.
- All experiments were performed on primary stroma of early passages (p3-p7) and exhibiting similar immunophenotype between the genotypes.
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The effects of various glucose conditions on PDGF-mediated migration of retinal AC.
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Transwell assay was performed with basal medium or medium containing PDGF-AA or PDGF-BB in lower compartment.
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The effects of various glucose conditions on PDGF-mediated migration of retinal AC.
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Transwell assay was performed with basal medium or medium containing PDGF-AA or PDGF-BB in lower compartment.
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In vitro differentiation of Nes-GFP+ cells
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LC differentiation For LC lineage differentiation, the Nes-GFP+ cells were replated in fresh differentiation-inducing medium containing phenol red-free DMEM/F12, 2% FCS, 10 ng/ml PDGF-BB , 1 ng/ml LH , 1 nM thyroid hormone , 70 ng/ml insulin-like growth factor 1 (IGF1), and ITS supplement , and they were incubated for 7 days, as previously described.
- Differentiation was subsequently confirmed by RT-PCR and immunostaining for LC lineage markers (antibodies shown in
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