A fluorometric assay for the measurement of endothelial, cell density in vitro

A fluorometric assay for determining endothelial cell numbers based on the endogenous enzyme acid phosphatase is described. In preliminary studies, th ree substrates-p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 2'-[2-benzthiazoyl ]-6'-hydroxy-benthiazole phosphate (AttoPhos(TM))-were compared with respect to their kinetic, optimum assay conditions, sensitivi ty, and detection limits. Only Attophos(TM) was found to have a high degree of sensitivity, reliability, and reproducibility for measuring both high a nd low cell numbers in the same plate. In subsequent experiments, assay con ditions were validated for measuring endothelial cell density in response t o basic fibroblast growth factor and fumagillin. Furthermore, the AttoPhos( TM) assay revealed a linear correlation between acid phosphatase activity a nd cell number in many cell types, including BALB/3T3, CHO-K1, A431, MCF7, 2008, SK-OV-5, T47-D, and OVCAR-3. This assay is potentially valuable for u se in many in vitro systems in which the quantitation of cell density and p roliferation is necessary. The practical advantages of AttoPhos TN assay fo r measuring endothelial cell numbers include (1) nonradioactivity, (2) simp licity, (3) economy (4) speed of assessment of proliferation of large numbe r of samples, and (5) amenability to high-throughput drug screening.