Analyzing Ca²⁺ Dynamics in Intact Epithelial Cells Using Spatially Limited Flash Photolysis

Abstract

The production of saliva by parotid acinar cells is stimulated by Ca²⁺ activation of Cl⁻ and K⁺ channels located in the apical plasma membrane of these polarized cells. Here we describe a paradigm for the focal photorelease of either Ca²⁺ or an inositol 1,4,5 trisphosphate (InsP₃) analog. The protocol is designed to be useful for investigating subcellular Ca²⁺ dynamics in polarized cells with minimal experimental intervention. Parotid acinar cells are loaded with cell-permeable versions of the caged precursors (NP-EGTA-AM or Ci-InsP₃/PM). Photolysis is accomplished using a spatially limited, focused diode laser, but the experiment can be readily modified to whole-field photolysis using a xenon flash lamp.