Gibco™ Dulbecco's Modified Eagle Medium (DMEM) low glucose supplemented with GlutaMAX™ and pyruvate, fetal bovine serum (FBS), and penicillin/streptomycin solution 10,000 units ml−1 (P/S), amphotericin B 250 μg ml−1, PBS, and 0.05% trypsin/EDTA was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Entactin-Collagen-Laminin (ECL) was purchased from Millipore Sigma (Burlington, MA, USA), and bovine skin collagen was purchased from Sigma–Aldrich (BioReagent). Dimethyl sulfoxide (DMSO), collagenase, and Cytodex®1 microcarrier were purchased from Sigma–Aldrich (Merck KGaA, Darmstadt, Germany). Ultroser™ G was purchased from PALL Corporation (NY, USA).
Bovine skin collagen
Manufactured by Merck Group
Sourced in United States
Bovine skin collagen is a type of extracellular matrix protein derived from the skin of cattle. It is a key structural component that provides support and strength to tissues. This product can be used as a raw material in various applications, such as cell culture and tissue engineering.
Automatically generated - may contain errors
Lab products found in correlation
Transwell insert, by Corning (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Thrombin, by Lonza (2 mentions)
Thrombin, by Merck Group (2 mentions)
Anti ve cadherin, by LifeSpan BioSciences (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Tnf α, by R&D Systems (2 mentions)
Ebm 2 media, by Lonza (2 mentions)
Lipopolysaccharides (lps), by Lonza (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Glutamax and pyruvate, by Thermo Fisher Scientific (1 mentions)
Cytodex 1 microcarrier, by Merck Group (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Ultroser g, by Pall Corporation (1 mentions)
4 protocols using bovine skin collagen
1
Cell Culture Reagents and Supplements
2
Monolayer Permeability and VE-Cadherin Assays
3
Monolayer Permeability and VE-Cadherin Assays
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Monolayer permeability was assessed as described previously49 (link). Briefly, transwell inserts (Corning, Corning, NY) were coated for 1 hour at 37°C with 0.1 mg/mL bovine skin collagen (Sigma), washed with water 2 times and rinsed with medium prior to plating cells. Cells (4×105 in 100 μl complete EGM-2 medium) were plated in the top chamber and 700 μl of medium was added to the bottom chamber 24 hours prior to the addition of fresh medium to both chambers, with or without 10 or 20 ng/mL BMP9 in the upper chamber (as indicated). After 30 minutes, 25 nM horseradish peroxidase was added to the top chamber, with or without 0.4ug/ml LPS (Sigma), 1U/mL thrombin (Sigma) or 50 ng/mL TNFα (R&D Systems). Medium (20μl) was collected from the lower chamber every 30 minutes for 2 hours. HRP content of the media samples was determined by measuring absorbance after the addition of 150μl of o-Phenylenediamine dihydrochloride buffer to each well of a 96-well plate.
For VE-cadherin assays, PAECs were cultured in EBM-2 media (Lonza) with 2% FBS for 24 hours with or without LPS, thrombin, TNFα and/or 10 ng/mL BMP9. Cells were then stained with FITC-conjugated rabbit polyclonal anti-VE-cadherin (cat #: LS-C40121, 1:100, Lifespan Biosciences, Seattle, WA).
For VE-cadherin assays, PAECs were cultured in EBM-2 media (Lonza) with 2% FBS for 24 hours with or without LPS, thrombin, TNFα and/or 10 ng/mL BMP9. Cells were then stained with FITC-conjugated rabbit polyclonal anti-VE-cadherin (cat #: LS-C40121, 1:100, Lifespan Biosciences, Seattle, WA).
4
Endothelial Permeability Assay Protocol
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Monolayer permeability was assessed as described previously.32 (link) Briefly, transwell inserts (Corning, Corning, NY) were coated for 1 h at
37℃ with 0.1 mg/ml bovine skin collagen (Sigma), washed twice with water and
rinsed with medium before plating cells. BOECs (400,000 cells in 100 µl Complete
EGM-2 medium) were plated in the top chamber and 700 µl of medium was added to
the bottom chamber 24 h before addition of fresh medium to both chambers, with
or without 5 ng/ml BMP9 or PTC124 (100 µM) in the upper chamber. After 30 min,
25 nM horseradish peroxidase (HRP) was added to the top chamber, with or without
0.4 µg/ml lipopolysaccharide (LPS) (Sigma). Medium (20 µl) was collected from
the lower chamber every 15 min for 60 min. The HRP content of this medium was
determined by measuring absorbance at 490 nm after the addition of 150 µl of
o-phenylenediamine dihydrochloride buffer to each well of a
96-well plate.
37℃ with 0.1 mg/ml bovine skin collagen (Sigma), washed twice with water and
rinsed with medium before plating cells. BOECs (400,000 cells in 100 µl Complete
EGM-2 medium) were plated in the top chamber and 700 µl of medium was added to
the bottom chamber 24 h before addition of fresh medium to both chambers, with
or without 5 ng/ml BMP9 or PTC124 (100 µM) in the upper chamber. After 30 min,
25 nM horseradish peroxidase (HRP) was added to the top chamber, with or without
0.4 µg/ml lipopolysaccharide (LPS) (Sigma). Medium (20 µl) was collected from
the lower chamber every 15 min for 60 min. The HRP content of this medium was
determined by measuring absorbance at 490 nm after the addition of 150 µl of
o-phenylenediamine dihydrochloride buffer to each well of a
96-well plate.
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