Mag-Fluo4 in T cells: Imaging of intra-organelle free Ca(2+) concentrations

Ca2+ signaling is a major signal transduction pathway involved in T cell activation, but also in apoptosis of T cells. Since T cells make use of several Ca2+-mobilizing second messengers, such as nicotinic acid adenine dinucleotide phosphate, d-myo-inositol 1,4,5-trisphosphate, and cyclic ADP-ribose, we intended to analyze luminal Ca2+ concentration upon cell activation. Mag-Fluo4/AM, a low-affinity Ca2+ dye known to localize to the endoplasmic reticular lumen in many cell types, showed superior brightness and bleaching stability, but, surprisingly, co-localized with mito-tracker, but not with ER-tracker in Jurkat T cells. Thus, we used Mag-Fluo4/AM to monitor the free luminal mitochondrial Ca2+ concentration ([Ca2+]mito) in these cells. Simultaneous analysis of the free cytosolic Ca2+ concentration ([Ca2+]i) and [Ca2+]mito upon cell stimulation revealed that Ca2+ signals in the majority of mitochondria were initiated at [Ca2+ ]i≥approx. 400 to 550nM. In primary murine CD4+ T cells, Mag-Fluo4 showed two different localization patterns: either co-localization with mito-tracker, as in Jurkat T cells, or with ER-tracker. Thus, in single primary murine CD4+ T cells, either decreases of [Ca2+ ]ER or increases of [Ca2+ ]mito were observed upon cell stimulation. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.