Measurement of cytosolic free calcium in mammalian cells with aequorin

Abstract

Measurement of cytosolic free calcium (Cai) in small mammalian cells has been achieved by incorporating into cells the photoprotein aequorin by hypoosmotic shock treatment (HOST). The method and the instrumentation necessary for monitoring Cai are described in detail. We present evidence that the aequorin-Ca light signal originates in the cytosol and that the cells subjected to the HOST procedure have a normal viability and functional integrity with respect to growth, respiration, membrane transport calcium metabolism, and hormone responsiveness. From 36 measurements made at 25 degrees C, the cytosolic free calcium of cultured kidney cells is estimated to be 60 nM assuming an intracellular free magnesium of 1 mM. Anoxia and metabolic inhibitors (10 mM cyanide) dramatically increase the cytosolic free calcium, and both effects are fully reversible.