Hi, I am working with epithelial cells from the bladder, to form an in vitro urothelial equivalent. I will like to test how permeable it is and whether it is similar to the native urotelium. Any ideas?
Barrier integrity is easily assessed by using HRP-dextran, polycarbonate filters (8.0 micrometer pore size), and plate reader. Add HRP-dextran to the upper layer of confulent monolayers on polycarbonate filter. After 60 minutes incubation, collect the medium of lower compartment, develop a color, and measure the absorbance by plate reader. If you want to know the details, please refer the manuscript "Alcohol Clin and Exp Res 2005 29(12) 2116-2122". This experiment is a simple method, need not expensive laboratory instruments, and can determine directly the cell permeability. Please try this methods.
Regrettablly, we did not do experiments using combination of both 0.5 micrometer pore size polycarbonate filetes and 40kDal HRP dextran. However, diameter of 40kDal protein is 2nm (similar size with endotoxin), and pore size of filter is 500 nm(=0.5micrometer), larger than that of 40kDal HRP. Therefore, theoretically, 40kDal HRP-dextran work using smaller filters. Good luck!!