A novel fluorescence-based cellular permeability assay.
Vascular permeability is a pathologic process in many disease states ranging from metastatic progression of malignancies to ischemia-reperfusion injury. In order to more precisely study tissue, and more specifically cell layer permeability, our goal was to create a fluorescence-based assay which could quantify permeability without radioactivity or electrical impedance measurements. Human aortic endothelial cells were grown in monolayer culture on Costar-Transwell clear polyester membrane 6-well cell culture inserts. After monolayer integrity was confirmed, vascular endothelial growth factor (VEGF(165)) at varying concentrations with a fixed concentration of yellow-green fluorescent 0.04 microm carboxylate-modified FluoSpheres microspheres were placed in the luminal chamber and incubated for 24 h. When stimulated with VEGF(165) at 20, 40, 80, and 100 ng/ml, this assay system was able to detect increases in trans-layer flux of 8.2+/-2.4%, 16.0+/-3.7%, 41.5+/-4.9%, and 58.6+/-10.1% for each concentration, respectively. This represents the first fluorescence-based permeability assay with the sensitivity to detect changes in the permeability of a cell layer to fluid flux independent of protein flux; as well as being simpler and safer than previous radioactive-and impedance-based permeability assays. With the application of this in vitro assay to a variety of pathologic conditions, both the dynamics and physiology relating to cellular permeability can be more fully investigated.
Chandra
Ankur
A
Section of Vascular and Endovascular Surgery, Department of Surgery, Univeristy of California, San Diego Medical Center, 200 West Arbor Drive, San Diego, CA 92103-8402, USA.
Barillas
Samuel
S
Suliman
Ahmed
A
Angle
Niren
N
D001703:Biophysics / Q000379:methods; D002199:Capillary Permeability / Q000187:drug effects / Q000502:physiology; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000502:physiology; D005456:Fluorescent Dyes; D006801:Humans; D008863:Microspheres; D008954:Models, Biological; D011994:Recombinant Proteins / Q000494:pharmacology; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
D005456:Fluorescent Dyes; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A