Imaging Initial Ca2+ Microdomains in Primary T Cells.

Local, sub-second Ca ²⁺ signals, termed Ca ²⁺ microdomains, are highly dynamic and short-lived Ca ²⁺ signals, which result in a global [Ca ²⁺ ]i elevation and might already determine the fate of a T cell. Upon T cell receptor activation, NAADP is formed rapidly, binding to NAADP binding proteins (HN1L/JPT2, LSM12) and their respective receptors (RyR1, TPC2) sitting on intracellular Ca ²⁺ stores, like the ER and lysosomes, and leading to subsequent release and elevation of [Ca ²⁺ ]i. To capture these fast and dynamically occurring Ca ²⁺ signals, we developed a high-resolution imaging technique using a combination of two Ca ²⁺ indicators, Fluo-4 AM and FuraRed AM. For postprocessing, an open-source, semi-automated Ca ²⁺ microdomain detection approach was developed based on the programming language Python. Using this workflow, we are able to reliably detect Ca ²⁺ microdomains on a subcellular level in primary murine and human T cells in high temporal and spatial resolution fluorescence videos. This method can also be applied to other cell types, like NK cells and murine neuronal cell lines.