T-cell calcium dynamics visualized in a ratiometric tdTomato-GCaMP6f transgenic reporter mouse

Calcium is an essential cellular messenger that regulates numerous functions in living organisms. Here, we describe development and characterization of 'Salsa6f', a fusion of GCaMP6f and tdTomato optimized for cell tracking while monitoring cytosolic Ca²⁺, and a transgenic Ca²⁺ reporter mouse with Salsa6f targeted to the Rosa26 locus for Cre-dependent expression in specific cell types. The development and function of T cells was unaffected in Cd4-Salsa6f mice. We describe Ca²⁺ signals reported by Salsa6f during T cell receptor activation in naive T cells, helper Th17 T cells and regulatory T cells, and Ca²⁺ signals mediated in T cells by an activator of mechanosensitive Piezo1 channels. Transgenic expression of Salsa6f enables ratiometric imaging of Ca²⁺ signals in complex tissue environments found in vivo. Two-photon imaging of migrating T cells in the steady-state lymph node revealed both cell-wide and localized sub-cellular Ca²⁺ transients ('sparkles') as cells migrate.