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In vitro endothelial permeability assay

MK Misun Kwak
CH Chansik Hong
JM Jongyun Myeong
EP Eunice Yon June Park
JJ Ju-Hong Jeon
IS Insuk So
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To measure permeability changes of endothelial cells in vitro, HUVECs were cultured to confluence on cell culture inserts (3.0 μm, BD Biosciences) placed in a 12-well plate. 5 × 105 HUVECs were seeded on each cell culture insert. In control samples, 500 μL of Evans blue dye (0.67 mg/mL) was added to the upper compartment to demonstrate that the endothelial layers were impermeable to this dye. Experimental wells were treated with siPKD1, siTRPC4 or ML204 for 48 hours, and 500 μL dye was added to the upper compartments. The liquid was collected from the lower wells after 10 minutes and measured by spectrophotometric absorbance at 570 nm.

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