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Published on: November 25, 2015
Phosphorimaging detection and quantitation for isotopic ion flux assays
1Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA. rfitch@indstate.edu
Abstract:
A 96-well-microplate-based ion flux method utilizing readily available autoradiographic phosphorimaging detection is described. Nicotinic acetylcholine receptor-mediated (22)Na 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 (86)Rb efflux produced data similar to data obtained with the (22)Na influx assay. However, assays for (45)Ca influx were not successful, although (45)Ca was readily detected and quantified. Voltage-gated sodium channel-mediated (22)Na 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 1,200 simultaneous samples with excellent sensitivity and is amenable for application to high-throughput screening.
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