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

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.