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. 2016 Oct 24;215(2):231–243. doi: 10.1083/jcb.201602001

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© 2016 Monaco et al.

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Figure 1. — TCR activation induces calcium transients in the cytoplasm and the nucleus in primary human T cells. (A) Confocal images showing the subcellular localization of the genetically encoded calcium indicators GCaMP3 (cytoplasm and nucleus), GCaMP3.NLS (nucleus), and GCaMP3.NES (cytoplasm). Bar, 5 µm. (B–D) Representative experiments showing calcium transients in human T cells transfected with the indicated calcium sensors and stimulated with CD3 antibodies. Anti–mouse IgG (black arrows) was added for antibody cross-linking, and calcium signals were measured for 9 min. Ionomycin (red arrows) and EGTA (blue arrows) were added to obtain the maximum (F_max) and minimum (F_min) signal, respectively. Calcium signals were analyzed by monitoring changes in GFP mean fluorescence as a function of time. The dot plots (left) are representative examples of at least four independent experiments (a.u., arbitrary units). Histograms (right) show mean GFP fluorescence (ΔF/F₀) from seven (GCaMP3), four (GCaMP3.NLS), and four (GCaMP3.NES) independent experiments. Error bars represent SEM.