Primary human aortic ECs (HAoECs; PCS-100-011) were purchased from American Type Culture Collection and cultured as before.27 (link) HAoECs were used at passages 3 to 5 for the experiments described in this study. Adam15 siRNA (Ambion; s13718, siRNA#16682) was used to knock down Adam15 in HAoECs, and scrambled siRNA (Ambion; 4390844) was used as control. In vitro EC permeability assay was performed using a vascular permeability assay kit according to the manufacturer’s instructions (EMD Millipore) as before.27 (link)
Vascular permeability assay kit
Manufactured by Merck Group
Sourced in United States
The Vascular Permeability Assay Kit is a laboratory instrument used to measure the permeability of vascular endothelial cells. It provides a quantitative assessment of endothelial barrier function.
Automatically generated - may contain errors
Lab products found in correlation
Scrambled sirna, by Thermo Fisher Scientific (1 mentions)
Haoecs pcs 100 011, by American Type Culture Collection (1 mentions)
Haoecs, by American Type Culture Collection (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Fitc conjugation kit fast lightning link, by Abcam (1 mentions)
Recombinant mouse il 1β, by Thermo Fisher Scientific (1 mentions)
Multimode reader, by Agilent Technologies (1 mentions)
Transwell polyester membrane insert, by Corning (1 mentions)
Millicell ers, by Merck Group (1 mentions)
8 protocols using vascular permeability assay kit
1
Culturing and Permeability Assay of Human Aortic Endothelial Cells
2
Vascular Permeability and Neutrophil Migration Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human microvascular endothelial HULEC-5a cells (ATCC, CRL-3244) were cultured in MCDB131 medium containing 10% FBS, 10 ng/mL epidermal growth factor (EGF), 1 µg/mL hydrocortisone and 10 mM glutamine (ThermoFisher Scientific, Waltham, MA, USA). The permeability of HULEC-5a cells was assessed using an in vitro Vascular Permeability Assay kit (EMD Millipore, Burlington, MA, USA). Briefly, HULEC-5a cells were seeded and then treated with various concentrations of S100A6 protein (R&D system) for 3 days to confluence in inserts. FITC-Dextran solution was then added to the monolayer of HULEC-5a cells and incubated for 1 h. The medium in the bottom well was collected and the FITC-Dextran was measured (excitation at 485 nm, emission at 535 nm). HULEC-5a cells were seeded and grown to confluence in an insert (pore size: 1 μm, EMD Millipore, Burlington, MA, USA). Freshly isolated human neutrophils (5 × 105 cells) were added to the inserts, and coculture was carried out for 12 h. The migratory neutrophils in the bottom well were then counted.
3
Vascular Permeability Assay of FK506 and TM
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The effects of FK506 and TM mutants on vascular permeability were measured by a vascular permeability assay kit (Millipore, Billerica, MA). Briefly, HUVECs were plated onto collagen-coated inserts and cultured for 72 h until confluence. After starvation for 24 h, cells were treated with TME5A/B/C (500 nM) or TME5 (30 nM) with or without FK506 (10 μg/ml) for 12 h. Then fluorescein isothiocyanate-dextran was added. The extent of permeability was determined by measuring the fluorescence of the plate well solution (excitation: 485 nm, emission: 535 nm).
4
Transendothelial Permeability Assay
5
Evaluating Endothelial Barrier Function
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Endothelial barrier function and integrity were observed through FITC-dextran clearance assay and TER assay. In the FITC-dextran clearance assay, CMECs were incubated 2 h with FITC-dextran (final concentration 1 mg/mL), washed with PBS, and analyzed using a fluorescent plate reader (Bio-Rad, USA). The TER assay was performed using the Vascular Permeability Assay Kit (ECM640; Millipore, USA).64 (link)
6
Assessing Lung Endothelial Permeability
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lung primary microvascular endothelial cells (Cell Biologics, C57-6011) were grown in phenol red–free endothelial cell medium (ScienCell), seeded in collagen-coated inserts, and permeability was determined using a vascular permeability assay kit according to the manufacturer’s instructions (Millipore). FITC-conjugated anti-COLV antibody, labeled using a FITC Conjugation Kit (Fast) - Lightning-Link (Abcam), was added to wells in the absence or presence of 50 ng/mL recombinant mouse IL-1β (Thermo Fisher Scientific) and fluorescence was red with a plate reader with filters appropriate for 485 nm and 535 nm excitation and emission, respectively.
7
Assessing Endothelial Cell Permeability
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
16HBE cells were cultured on transwell inserts placed in a 24-well plate until 100% confluence was reached; then, they were infected with EV-A71 or CV-A16. Following completion of the treatment, the permeability of cell monolayers was determined with an in vitro Vascular Permeability Assay kit (Millipore, USA). Briefly, the medium was carefully removed from the transwell insert, and 150 μl of FITC-Dextran working solution was added to the apical surface of 16HBE cells. The 24-well plate was protected from light and incubated for 20 min at room temperature. Next, the medium from each well of the receiver tray was thoroughly mixed, and 100 μl was transferred to the wells of a black 96-well opaque plate for fluorescence measurement. The fluorescein dye was measured in a multi-mode reader (Biotek, USA) at 480/530 nm (excitation/emission). The data are representative of at least three experiments.
8
Transendothelial Permeability and Resistance Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transendothelial permeability to dextran was performed as previously described [30] according to manufacturer's instructions using labeled tracer flux across confluent RIMECs grown on the Transwell inserts with polycarbonate filters (Vascular Permeability Assay Kit, Millipore). In brief, FITC-labeled dextran (∼60 kDa) was added to the upper chamber. At indicated time points (0, 2, 4, 6, 8 hr), 50 μl of media were collected from the lower chamber. The amount of FITC-labeled dextran filtrating into the lower chamber was determined by the fluorescence spectrophotometer. The experimental data were expressed as arbitrary fluorescence units.
Transendothelial electrical resistance (TEER) 2 x 10 5 RIMECs were cultured to confluence on a Transwell polyester membrane insert (0.4 μm pore size and 6.5 mm in diameter) (Corning, USA) on 24-well culture plates and were serum-starved overnight. At indicated time points (0, 2, 4, 6, 8 hr), the TEER was measured by using Millicell-ERS (Millipore, Germany) as previously described [31] [32] [33] . The TEER were detected and compared as the percent change from corresponding baseline values. Each experiment was repeated three times.
Transendothelial electrical resistance (TEER) 2 x 10 5 RIMECs were cultured to confluence on a Transwell polyester membrane insert (0.4 μm pore size and 6.5 mm in diameter) (Corning, USA) on 24-well culture plates and were serum-starved overnight. At indicated time points (0, 2, 4, 6, 8 hr), the TEER was measured by using Millicell-ERS (Millipore, Germany) as previously described [31] [32] [33] . The TEER were detected and compared as the percent change from corresponding baseline values. Each experiment was repeated three times.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!