Continuous Assay Method for Measuring Receptor- and Ion Channel-Mediated Calcium Response and Subsequent Gene Expression

摘要

To measure a receptor-mediated increase in intracellular calcium concentration, use is made of a calcium-responding luminescent protein localized in the cytoplasm or mitochondria and a reporter gene having a promoter responding to the second messenger in cells and Renilla-origin luciferase attached to the downstream thereof. By using a coelenterazine derivative, which can exist in the cells in a stable state over a long time, as the substrate of these proteins, a transient increase in the intracellular calcium concentration observed within several seconds is measured by using the calcium-responding luminescent protein. Subsequently, the cells are allowed to survive and cultured for several hours as such without adding a fresh substrate. During this period, an increase/decrease in the gene expression is continuously monitored by the reporter assay method and it is measured and evaluated, if necessary, whether or not the inhibition of the activity is caused by cytotoxicity.