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.