Label-Free Profiling of Endogenous Receptor Responses in Primary Isolated Cardiac Cells

Abstract

Label-free detection systems have been available and utilized for several years in the pharmacological exploration of receptor responses to ligand stimulation. While a vast majority of studies have investigated signaling responses in cell lines stably expressing a receptor of interest, relatively fewer studies have used label-free technology to examine endogenous receptor responses in primary cells with more physiologic relevance. The exploration of cardiac receptor biology is fraught with challenges, primarily stemming from the difficulty in maintaining isolated adult cardiac myocytes under cell culture conditions in sufficient quantity and for extended periods that may be required for various assays. However, isolated rat neonatal cardiac myocytes and fibroblasts offer an alternate approach that allows in vitro investigation of primary cardiac cells for several days to weeks. For use with label-free technologies, primary cardiac cells provide a unique opportunity to explore the impact of various ligands on endogenous cardiac receptor responses, which in the case of myocytes may also be directly applicable to regulation of contractile function. In this chapter we provide a detailed methodology for the isolation of primary cardiac myocytes and fibroblasts and discuss their use in label-free assays, using the Epic BT resonant waveguide grating biosensor system as an example, with a particular consideration toward cardiac cell density and phenotypic modulation of cells in culture.