Measuring Intranuclear and Nuclear Envelope [Ca2+] vs. Cytosolic [Ca2+]
Published Web Locationhttp://link.springer.com/protocol/10.1007/978-1-4939-1755-6_12
Nuclear Ca(2+) regulates key cellular processes, including gene expression, apoptosis, assembly of the nuclear envelope, and nucleocytoplasmic transport. Quantification of subcellularly resolved Ca(2+) signals is, therefore, essential for understanding physiological and pathological processes in various cell types. However, the properties of commonly used Ca(2+)-fluorescent indicators in intracellular compartments may differ, thus affecting the translation of Ca(2+)-dependent fluorescence changes into quantitative changes of Ca(2+) concentration. Here, we describe technical approaches for reliable subcellular quantification of [Ca(2+)] in the cytoplasm vs. the nucleus and the nuclear envelope by in situ calibration of fluorescein-derived fluorescent indicators Fluo-4 and Fluo-5N.