Live-Cell Visualization of Calcium Flux in Vibratome-Cut Thick Sections of Viable Tumor Tissue.

ABSTRACT

This unit outlines a live-cell imaging approach developed for visualization of intracellular calcium flux in human parathyroid tumors following stimulation of the calcium-sensing receptor (CASR), a class C G protein-coupled receptor (GPCR). The primary assay readout, intracellular calcium release induced by activation of the inositol triphosphate receptor, is potentially generalizable to multiple other GPCR signaling events that utilize this common downstream signal transduction pathway. Advantages of the approach include (1) preservation of native tissue context and positional information, allowing direct visualization of intratumoral functional heterogeneity; (2) quantitative documentation of reactivity to a physiological stimulus in an experimentally tractable ex vivo system; and (3) generation of a dynamic, functional classifier of tumor biochemical behavior to augment static marker assessment. The technical steps are performed in three sequential phases (1) viable tissue sectioning; (2) fluorophore loading and tissue immobilization; and (3) live-cell confocal microscopy. This versatile method provides a straightforward platform for functional characterization of human tumors. © 2017 by John Wiley & Sons, Inc.