Intracellular Ca2+ can be conveniently monitored by sensitive Ca2+ fluorescent dyes in live cells. The Gαq involved lipid signaling pathways and, thus, can be studied by intracellular Ca2+ imaging. Here we describe the protocols to measure intracellular Ca2+ for studying PEG2-EP1 activity in esophageal smooth muscle cells. The ratiometric Fura-2 imaging provides quantitative data, and the Fluo-4 confocal microscopic imaging has high-spatial resolution.

Live-cell imaging can reveal dynamic and multimodal cell signaling by monitoring calcium flux. Spatiotemporal changes in Ca2+ concentrations instigate specific downstream processes and by categorizing these events, we can examine the language cells use to communicate both to themselves and with each other. Thus, calcium imaging is an understandably popular and versatile technique that relies on high-resolution optical data as measured by fluorescence intensity. This is executed with relative ease on adherent cells, as changes in fluorescence intensity can be monitored over time in fixed regions of interest. However, perfusion of non-adherent or mildly adherent cells leads to their mechanical displacement thereby hindering the spatial resolution of fluorescence intensity changes through time. Here we provide details of a simple and cost-effective protocol using gelatin to prevent cell dislodgement during the solution exchanges that occur during recording.

Stimulus-secretion coupling in pancreatic β-cells requires Ca2+ influx through voltage-dependent Ca2+ channels, whose activity is controlled by the plasma membrane potential (V m). Here, we present a method of measuring fluctuations in the β-cell V m and Ca2+ influx simultaneously, which provides valuable information about the ionic signaling mechanisms that underlie insulin secretion. This chapter describes the use of perforated patch clamp electrophysiology on cells loaded with a fluorescent intracellular Ca2+ indicator, which permits the stable recording conditions needed to monitor the V m and Ca2+ influx in β-cells. Moreover, this chapter describes the protocols necessary for the preparation of mouse and human islet cells for the simultaneous recording of V m and Ca2+ as well as determining the specific islet cell type assessed in each experiment.