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In vitro endothelial permeability assay

KK Kayo Kunimura
SM Satomi Miki
MT Miyuki Takashima
JS Jun-ichiro Suzuki
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Endothelial permeability was measured by using in vitro vascular permeability assay kit (Merck Millipore). HUVECs were seeded at 1.5 × 103 cells/well on transwell inserts and cultured in a receiver plate. After grown to be confluent, HUVECs were stimulated with 50 ng/mL TNF-α or 1 U/mL thrombin in the absence or presence of test substances (1–4 mg/mL AGE, 75–300 μM S1PC, 300 μM SAC or 300 μM SAMC) for 24 h. In the case of the study using the inhibitors of RhoA and Rac signaling molecules, HUVECs were pre-treated with Y-27632 (a ROCK inhibitor, 10 μM) or PD98059 (a MEK1 inhibitor, 30 μM) for 1 h before the TNF-α stimulation. Then, FITC-dextran was added on each transwell insert and the extent of permeability was determined by measuring the fluorescence of each receiver plate solution. The fluorescence intensity was quantified with EnVision plate reader (PerkinElmer, Waltham, MA, USA) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

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