Fibronectin-coated (5 μg/ml, BD Biosciences, Franklin, Lakes, NJ, USA) polyacrylamide gels were prepared on glass slides (Fisher, Waltham, MA, USA) as previously described22 (link). Two ratios of acrylamide to bis-acrylamide (Fisher), 50:1 and 12.5:1, were used to make gels with Young’s moduli of 1 kPa and 308 kPa, respectively. The Young’s moduli of the polyacrylamide gels were measured using a rheometer as previously described22 (link).
Fibronectin coated
Manufactured by BD
Sourced in United States
Fibronectin-coated lab equipment is a specialized surface coating designed to facilitate cell attachment and growth in in vitro cell culture applications. Fibronectin is an extracellular matrix protein that promotes cell adhesion, spreading, and proliferation. The fibronectin coating provides a more natural and biomimetic substrate for various cell types, enhancing their ability to adhere, spread, and thrive in laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Magnetic column, by Miltenyi Biotec (2 mentions)
Vegf165, by R&D Systems (2 mentions)
Chir99021, by STEMCELL (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Glass slide, by Thermo Fisher Scientific (1 mentions)
Acrylamide, by Thermo Fisher Scientific (1 mentions)
Bis acrylamide, by Thermo Fisher Scientific (1 mentions)
Alexa 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Glass slide, by BD (1 mentions)
Fluoromount, by Merck Group (1 mentions)
Ebm 2 medium, by Lonza (1 mentions)
Mtesr1 medium, by STEMCELL (1 mentions)
Anti pe microbeads, by Miltenyi Biotec (1 mentions)
Stempro accutase, by Thermo Fisher Scientific (1 mentions)
Insulin like growth factor 1, by Thermo Fisher Scientific (1 mentions)
Vascular endothelial growth factor (vegf), by Thermo Fisher Scientific (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
7 protocols using fibronectin coated
1
Polyacrylamide Gel Fabrication and Characterization
2
Immunofluorescence Microscopy of Cells
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Cells on fibronectin-coated (10 µg/ml) glass slides (BD Biosciences) were fixed in 4% paraformaldehyde (PFA), permeabilised with 0.2% Triton X-100 and blocked (1 h, room temperature) in phosphate-buffered saline (PBS)/0.2% bovine serum albumin (BSA), prior to incubation (30 min, 37°C) with appropriate antibodies in PBS/0.2% BSA, and Alexa-conjugated secondary antibodies (1 h, room temperature). In some cases cells were stained with fluorescently conjugated antibodies in culture media prior to fixation. Alternatively, actin was stained in permeabilised cells with Alexa488-phalloidin (1 µg/ml; Molecular Probes). For cell rounding assays, IIIA4 mAb (1.5 or 3.0 µg/ml), cross-linked with anti-mouse IgG (Jackson ImmunoResearchs), was added to cells 10 min prior to analysis. Coverslips were mounted onto microscope slides with Fluoromount (Sigma). Fluorescence images were taken on a Leica SP5 microscope and analysed using AnalySIS software (Soft Imaging System, Germany).
3
Derivation and Culture of Isogenic Trisomic and Disomic iPSCs
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Isogenic disomic/trisomic iPSCs (4C4-Disomic/4C4-Trisomic) were kindly provided by Dr. Stuart Orkin. H9 (WA09) was purchased from WiCell. Isogenic disomic iPSCs (isoDS-iPSCs), trisomic iPSCs (DS-iPSCs), and disomic SR2-iPSCs were derived as previously described in12 (link),13 (link). Prior to differentiation, the cells were maintained on Matrigel-coated culture dishes in mTeSR1 medium (STEMCELL Technologies). Differentiation was induced via addition of CHIR99021 (STEMCELL Technologies). Endothelial differentiation was also supplemented with vascular endothelial growth factor (VEGF165) (R&D Systems). On day 4, the differentiated ECs were isolated by immuno-selection of CD31+CD144+ cells via a magnetic column (Miltenyi Biotec). Following this, the SR2 and DSV-iECs were grown on fibronectin-coated (10 μg/mL) (BD Biosciences) plates and cultured in VascuLife EnGS medium (LifeLine) at 37 °C and 5% CO2. Mesodermal progenitors were isolated by immuno-selection of CD73 + cells and cultured in EBM 2 medium (Lonza).
4
Isolation and Characterization of Primary Human Cytotrophoblasts
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Primary human CTBs were isolated from first trimester placentae according to a modified protocol of Tarrade et al. [21] (link), as previously published [18,20,22] . Purified trophoblasts were seeded onto fibronectin-coated (20 μg/ml; BD Biosciences, Franklin Lakes, NJ) wells at a density of 5 × 105 cells/24-well. To isolate CTB subtypes, magnetic bead sorting (MACS) was used to separate proliferative, EGFR+ CTBs from differentiated, HLA-G+ CTBs. EGFR-PE antibody (Santa Cruz Biotechnology, Dallas, TX) or HLA-G-PE antibody (Exbio, Praha, Czech Republic) and anti-PE micro-beads (Miltenyi Biotec, Bergisch Gladbach, Germany) were utilised. SGHPL-5 cells were cultivated in DMEM/Ham's F12, supplemented with 10% FCS and 0.05 mg/ml gentamicin as published [23] (link).
5
Endothelial Cell Proliferation Assay
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30,000 control and trisomic endothelial cells were seeded per well onto fibronectin-coated (10 μg/mL) (BD Biosciences) 6-well plates. The cells were cultured in VascuLife EnGS medium (LifeLine) containing varying VEGF concentrations (0.5 ng/mL, 2 ng/mL, 20 ng/mL). When one of the cell lines reached confluence, all cells for a particular VEGF concentration were harvested with StemPro Accutase (ThermoFisher Scientific) and a cell count was performed.
6
Endothelial Differentiation of Stem Cells
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Endothelial differentiation of DSV-iPSCs, isoDSV-iPSCs, H9-ESCs, and SR2-iPSCs was induced via the addition of CHIR99021 (STEMCELL Technologies) and vascular endothelial growth factor (VEGF165) (R&D Systems) to the culture medium. On day 4, the differentiated iECs were isolated by immuno-selection of CD31+CD144+ cells via a magnetic column (Miltenyi Biotec). Following this, the iECs were grown on fibronectin-coated (10 μg/mL) (BD Biosciences) plates and cultured in VascuLife EnGS medium (LifeLine) at 37°C and 5% CO2.
7
Isolation and Culture of Bone Marrow-Derived Endothelial Progenitor Cells
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The isolation and culture of BM-EPCs were performed as described11 (link). Rat bone marrow cells were obtained by flushing femurs and humeri with
Dulbecco’s modified Eagle medium (DMEM)/F12 medium (Gibco, New York, NY, USA).
BMSCs were then isolated by density gradient centrifugation with the Histopaque
1077 (Sigma-Aldrich, St. Louis, MO, USA) from bone marrow cells. After washing
with red blood cell lysis buffer, bone marrow mononuclear cells were seeded into
culture flasks in DMEM/F12 medium supplemented with 10% fetal bovine serum
(Gibco). After 24 h, the plastic-adherent cells were removed, and the
nonadherent cells were collected, washed, and replated into fibronectin-coated
(10 μg/ml; BD Biosciences, San Jose, CA, USA) culture flasks with the inducing
medium containing DMEM/F12 medium supplemented with 10% fetal bovine serum, 20
ng/ml VEGF, 5 ng/ml basic fibroblast growth factor, 5 ng/ml epidermal growth
factor, 10 ng/ml insulin-like growth factor-1 (Peprotech, New Jersey, NJ, USA),
and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin).
Dulbecco’s modified Eagle medium (DMEM)/F12 medium (Gibco, New York, NY, USA).
BMSCs were then isolated by density gradient centrifugation with the Histopaque
1077 (Sigma-Aldrich, St. Louis, MO, USA) from bone marrow cells. After washing
with red blood cell lysis buffer, bone marrow mononuclear cells were seeded into
culture flasks in DMEM/F12 medium supplemented with 10% fetal bovine serum
(Gibco). After 24 h, the plastic-adherent cells were removed, and the
nonadherent cells were collected, washed, and replated into fibronectin-coated
(10 μg/ml; BD Biosciences, San Jose, CA, USA) culture flasks with the inducing
medium containing DMEM/F12 medium supplemented with 10% fetal bovine serum, 20
ng/ml VEGF, 5 ng/ml basic fibroblast growth factor, 5 ng/ml epidermal growth
factor, 10 ng/ml insulin-like growth factor-1 (Peprotech, New Jersey, NJ, USA),
and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin).
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