High throughput fluorescence assays for the measurement of mitochondrial activity in intact human neuroblastoma cells.
The mitochondrial permeability transition event is implicated in the activation phase of apoptosis and necrosis, and is therefore postulated to play a role in many disease states. Mitochondrial permeability transition is therefore of increasing pharmaceutical interest. Drug discovery requires the rapid screening of compound libraries to identify functionally active ligands. We report the development of two fluorescence-based approaches for screening compound libraries for effects on mitochondrial function. These assays use the fluorometric imaging plate reader in 96-well format, and two commercially available dyes: JC-1 and calcein-AM. We show here that a JC-1 assay proved highly amenable to HTS implementation. By combining this with a calcein-based assay, these approaches gave complementary information: JC-1 facilitates the discovery of modulators of mitochondrial polarization from a library of approximately 100,000 compounds screened at 8 microM, and the calcein assay identifies permeability transition pore-specific inhibitors.
Woollacott
A J
AJ
Department of Biochemistry, Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Harlow, Essex, UK.
Simpson
P B
PB
D001562:Benzimidazoles; D002232:Carbocyanines; D005452:Fluoresceins; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000502:physiology*; D009447:Neuroblastoma / Q000473:pathology* / Q000648:ultrastructure; D010539:Permeability; D014407:Tumor Cells, Cultured
D001562:Benzimidazoles; D002232:Carbocyanines; D005452:Fluoresceins; D005456:Fluorescent Dyes; C085925:calcein AM; C068624:5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine