Measuring intranuclear and nuclear envelope [ca2+] vs. cytosolic [ca2+]

Abstract

Nuclear Ca2+ regulates key cellular processes, including gene expression, apoptosis, assembly of the nuclear envelope, and nucleocytoplasmic transport. Quantifi cation of subcellularly resolved Ca2+ signals is, therefore, essential for understanding physiological and pathological processes in various cell types. However, the properties of commonly used Ca2+ -fl uorescent indicators in intracellular compartments may differ, thus affecting the translation of Ca2+ -dependent fl uorescence changes into quantitative changes of Ca2+ concentration. Here, we describe technical approaches for reliable subcellular quantifi cation of [Ca2+] in the cytoplasm vs. the nucleus and the nuclear envelope by in situ calibration of fl uorescein-derived fl uorescent indicators Fluo-4 and Fluo-5N.