Measurement of intracellular Ca2+ concentration in single cells using ratiometric calcium dyes

Abstract

Measurement of intracellular Ca2+concentration ([Ca2+]i) is useful to study the upstream and downstream events of Ca2+ signaling. Ca2+-binding proteins including EF-hand-containing proteins are important downstream effector molecules after an increase of [Ca2+]i. Conversely, these proteins can also act as key modulators for regulation of [Ca2+]i by sensing the Ca2+ levels in the intracellular organelles and cytoplasm. Here we describe a single-cell Ca2+ imaging technique that was used to measure the intracellular Ca2+ levels to examine the function of Ca2+-binding proteins, STIM1 and Calcium release-activated Calcium channel regulator 2A (CRACR2A), using ratiometric Ca2+ dye Fura-2 in adherent and non-adherent cells.