Phosphorimaging detection and quantitation for isotopic ion flux assays

A 96-well-microplate-based ion flux method utilizing readily available autoradiographic phosphorimaging detection is described. Nicotinic acetylcholine receptor-mediated 22Na influx in four cultured cell lines provided satisfactory concentration-response data for epibatidine and several other nicotinic agonists. The data were consistent with data obtained using standard 6-well assays. Assays for nicotinic-receptor-mediated 86Rb efflux produced data similar to data obtained with the 22Na influx assay. However, assays for 45Ca influx were not successful, although 45Ca was readily detected and quantified. Voltage-gated sodium channel-mediated 22Na influx in a neuroblastoma cell line allowed assay of the effects of such sodium channel activators as batrachotoxin and a pumiliotoxin B/scorpion venom combination. Phosphorimaging detection allows for reliable beta counting of up to 1200 simultaneous samples with excellent sensitivity and is amenable for application to high-throughput screening.