Matrigel (0.2 mL; BD Biosciences) was added to each well of a 24-well tissue-culture plate and allowed to solidify at 37°C for at least 30 minutes. Following gelation, 0.2 mL of a cell suspension containing 1 × 105 iPSC ECs in Growth Medium was placed on top of the Matrigel. The cultures were incubated at 37°C, 5% CO2, and observed at 24, 48, and 72 hours for observation of cellular formation into capillary-like structures. Ulex staining: Matrigel tubes were stained with the lectin Ulex europaeus (Sigma) after 24 hours to confirm the presence of human endothelial cells in the vessel-like formations. Briefly, Growth Medium was removed from the Matrigel layer and tubes were incubated with 10 µg/ml Ulex for 30 minutes at room temperature followed by imaging.
Ulex europaeus
Manufactured by Merck Group
Sourced in United States
Ulex europaeus is a laboratory product that serves as a lectin reagent. It is derived from the plant species Ulex europaeus, commonly known as gorse or furze. The core function of this product is to act as a binding agent for specific carbohydrate structures, which can be used in various biochemical and cell biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (1 mentions)
Matrigel, by Merck Group (1 mentions)
Dii ac ldl, by Biomedical Technologies (1 mentions)
Human fibronectin, by Merck Group (1 mentions)
Rm2145, by Leica (1 mentions)
Vegfr2, by Santa Cruz Biotechnology (1 mentions)
Uea 1, by Merck Group (1 mentions)
Epac1 sirna, by Horizon Discovery (1 mentions)
Hiperfect, by Qiagen (1 mentions)
5 protocols using ulex europaeus
1
Matrigel-based Angiogenesis Assay for iPSC-derived ECs
2
Isolation and Characterization of EPCs
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EPCs were prepared as previously described. [10] (link) Peripheral Blood Mononuclear Cells (PBMCs) were isolated from peripheral blood of healthy human volunteers as described by Sun et al by density-gradient centrifugation with Ficoll. Immediately after isolation, total MNCs (5*106 cells/mL medium) were plated on culture dishes coated with human fibronectin (Sigma) and maintained in endothelial basal medium (EBM, CellSystems) supplemented with EGM SingleQuots, VEGF (10 ng/mL) and 20% fetal bovine serum. The medium was replaced every 3 days. Adherent cells on 8 days of culture were stained by acetylated LDL and were labeled with DiI (DiI-acLDL, Biomedical Technologies) and fluorescein isothiocyanate (FITC)-labeled lectin from ulex europaeus (Sigma). Double-positive cells for DiI-acLDL and FITC-labeled lectin were identified as EPCs, as reported previously. All human researches were conducted in Changzheng Hospital. All procedure were permitted by the human volunteers with written consent and approved by the Committee on the Ethics of human Experiments of Changzheng Hospital.
3
Fetal CDH Lung and Heart Morphology
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Fetuses were harvested on the 30th day of pregnancy as described. The neonates
were weighed (body weight) and an intramuscular dose of 1 mL of
ketamine/xylazine was applied. The abdomen was opened to confirm the presence
and size of CDH. After that, the lungs and the heart were removed and weighed
(total lung weight, left lung weight, right lung weight, and heart weight),
being then immersed in 10% formaldehyde for preservation (Figure 1 ).
Samples were then dehydrated in increasing alcohol concentrations, cleared with
xylene, and embedded in paraffin. Blocks were sectioned with a microtome (Leica
RM 2145, Leica, Germany) into 5-µm sections, then mounted on histological
slides.
For immunohistochemistry, slides were dewaxed with xylene for 5 min and
rehydrated with ethanol. The slides were incubated overnight with primary
antibodies diluted in 5% bovine serum albumin: VEGFR2 (Santa Cruz Biotechnology,
USA), Ki-67 (Sigma-Aldrich, USA), and lectin Ulex europaeus(Sigma-Aldrich).
were weighed (body weight) and an intramuscular dose of 1 mL of
ketamine/xylazine was applied. The abdomen was opened to confirm the presence
and size of CDH. After that, the lungs and the heart were removed and weighed
(total lung weight, left lung weight, right lung weight, and heart weight),
being then immersed in 10% formaldehyde for preservation (
Samples were then dehydrated in increasing alcohol concentrations, cleared with
xylene, and embedded in paraffin. Blocks were sectioned with a microtome (Leica
RM 2145, Leica, Germany) into 5-µm sections, then mounted on histological
slides.
For immunohistochemistry, slides were dewaxed with xylene for 5 min and
rehydrated with ethanol. The slides were incubated overnight with primary
antibodies diluted in 5% bovine serum albumin: VEGFR2 (Santa Cruz Biotechnology,
USA), Ki-67 (Sigma-Aldrich, USA), and lectin Ulex europaeus(Sigma-Aldrich).
4
Mucus Layer Characterization in Caco-2 and HT29-MTX Co-Culture
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Human intestinal carcinoma epithelial (Caco-2 clone C2BBe1) and mucus-producing (HT29-MTX) cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% heat-inactivated fetal bovine serum (Atlanta Biologics), 1% GlutaMax™, and 1% Penicillin/Streptomycin. Caco-2 (passage 55–65) and HT29-MTX (passage 35–45) cells were seeded in 9:1 ratio on tissue culture treated 24 well plates at a density of 105 cells/cm2. After 3 weeks, cells were exposed to phosphate buffered saline (PBS) control, 1% CMC in PBS, or 0.5% Tween in PBS for 1 hr at 37 °C. Tween concentration was reduced to 0.5% to minimize cytotoxicity since exposure to 1% Tween for 1 hr resulted in cell death. After 1 hr exposure, cells were washed with phosphate buffered saline and stained with LIVE/DEAD™ solution (Molecular Probes™). The mucus layer was also stained for 1 hr at 37 °C with 1 mg/mL wheat germ agglutinin (WGA, Molecular Probes™) for sialic acid and N-acetylglucosaminyl, as well as 1 mg/mL lectin from Ulex europaeus (Sigma) for L-fucose. Confocal z-stacks of the mucus layer were obtained at 10× with the same imaging parameters used for each sample from three separate experiments. ImageJ was used to quantify intensity of fucose and sialic acid stains in the maximum intensity z-stack projection. Average values ± standard deviation are reported.
5
Angiogenesis Assay Using Collagen Matrices
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Angiogenesis assay was performed as previously described (59 (link)). Briefly, collagen/media solution was prepared on ice and mixed with HUVECs (1 × 106/ml) and/or HDFNs (5 × 105/ml). Subsequently, 30 μl of collagen/cell mixture was spotted onto a 5-mm woven nylon mesh ring (Tetko Inc., Elmford, NY). After the collagen/cell mixture was polymerized at 37°C, samples were transferred to a 96-well flat-bottom culture plate with media. The culture media consisted of EBM-2 supplemented with all “bullet kit” components except FBS, VEGF, and basic FGF. This basic media was supplemented with 1% FBS and VEGF (30 ng/ml). For Epac inhibition, 2.5 μM ESI-09 was added to the culture media, which was refreshed every day. For coculture assay, HUVECs were transiently transfected using HiPerFect (QIAGEN, Valencia, CA) with Epac1 siRNA (M007676-01-0005, Dharmacon, Lafayette, CO). On day 5, collagen-embedded cells were fixed in 4% PFA and stained with tetramethyl rhodamine isothiocyanate–labeled lectin (10 μg/ml; Ulex europaeus, UEA-I, Sigma-Aldrich) overnight. Images were taken by a Zeiss LSM confocal microscope.
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