Identification of gap junction blockers using automated fluorescence microscopy imaging.
Gap junctions coordinate electrical signals and facilitate metabolic synchronization between cells. In this study, the authors have developed a novel assay for the identification of gap junction blockers using fluorescence microscopy imaging-based high-content screening technology. In the assay, the communication between neighboring cells through gap junctions was measured by following the redistribution of a fluorescent marker. The movement of calcein dye from dye-loaded donor cells to dye-free acceptor cells through gap junctions overexpressed on cell surface membranes was monitored using automated fluorescence microscopy imaging in a high-throughput compatible format. The fluorescence imaging technology consisted of automated focusing, image acquisition, image processing, and data mining. The authors have successfully performed a high-throughput screening of a 486,000- compound program with this assay, and they were able to identify false positives without additional experiments. Selective and pharmacologically interesting compounds were identified for further optimization.
Li
Zhuyin
Z
Lead Discovery Technology, Lead Generation, Aventis Pharmaceutical, Bridgewater, NJ 08807, USA. julie.li@aventis.com
Yan
Yongping
Y
Powers
Elaine A
EA
Ying
Xiaoyou
X
Janjua
Khurram
K
Garyantes
Tina
T
Baron
Bruce
B
D000818:Animals; D001331:Automation; D002450:Cell Communication; D002455:Cell Division / Q000502:physiology; D016543:Central Nervous System Neoplasms / Q000188:drug therapy / Q000473:pathology; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005189:False Positive Reactions; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D017629:Gap Junctions / Q000187:drug effects*; D005910:Glioma / Q000188:drug therapy / Q000473:pathology; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008469:Meclofenamic Acid / Q000494:pharmacology; D008856:Microscopy, Fluorescence / Q000379:methods*; D051381:Rats; D012680:Sensitivity and Specificity; D013997:Time Factors; D014407:Tumor Cells, Cultured
D005452:Fluoresceins; D005456:Fluorescent Dyes; D008469:Meclofenamic Acid; C007740:fluorexon