Calcium Imaging in T Lymphocytes: a Protocol for Use with Genetically Encoded or Chemical Ca2+ Indicators.

Elevations in cytosolic calcium (Ca2+) drive a wide array of immune cell functions, including cytokine production, gene expression, and cell motility. Live-cell imaging of cells loaded with ratiometric chemical Ca2+ indicators remains the gold standard for visualization and quantification of intracellular Ca2+ signals; ratiometric imaging can be accomplished with dyes such as Fura-2, the combination of Fluo-4 and Fura-Red, or, alternatively, by expressing genetically-encoded Ca2+ indicators (GECI) such as GCaMPs. Here, we describe a detailed protocol for Ca2+ imaging of T cells in vitro using genetically encoded or chemical indicators that can also be applied to a wide variety of cell types. The protocol addresses the challenge of facilitating T cell attachment on various substrates prepared on glass-bottom dishes to enable T cell imaging on an inverted microscope. The protocol also emphasizes cell preparation steps that ensure optimal cell viability - an essential requirement for recording dynamic changes in cytosolic Ca2+ levels - and that ensure reproducibility between multiple samples. Finally, we describe a simple algorithm to analyze single-cell Ca2+ signals over time using Fiji (ImageJ) software

Ion channel mediated mechanotransduction in immune cells

The immune system performs critical functions to defend against invading pathogens and maintain tissue homeostasis. Immune cells reside within or are recruited to a host of mechanically active tissues throughout the body and, as a result, are exposed to varying types and degrees of mechanical stimuli. Despite their abundance in such tissues, the role of mechanical stimuli in influencing immune cell function and the molecular mechanisms responsible for mechanics-mediated changes are still poorly understood. The recent emergence of mechanically-gated ion channels, particularly Piezo1, has provided an exciting avenue of research within the fields of mechanobiology and immunology. Numerous studies have identified roles for mechanically-gated ion channels in mechanotransduction within various different cell types, with a few recent studies in immune cells. These initial studies provide strong evidence that mechanically-gated ion channels play pivotal roles in regulating the immune system. In this review, we discuss characteristics of ion channel mediated force transduction, review the current techniques used to quantify and visualize ion channel activity in response to mechanical stimuli, and finally we provide an overview of recent studies examining the role of mechanically-gated ion channels in modulating immune cell function

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