Multianalyte Single-Cell Analysis with Multiple Cell Lines Using a Fiber-Optic Array

A single-cell drug screening method is described that produces rich single-cell data and discriminates between single-cell responses from clonal populations stimulated with different agonists. Ligand-induced receptor activation is commonly detected by observing intracellular Ca2+ oscillations using high-throughput screening (HTS) methods. In most cases, HTS results in an average signal from several cells and is not sensitive enough to enable the identification of population outliers or population variance. In order to obtain this information, many individual cells must be analyzed simultaneously. We have developed a novel system using a specialized fiber-optic platform and have combined it with statistical analysis, to simultaneously analyze the dynamics of Ca2+ oscillations in a large number of single cells. Mammalian cells ectopically expressing different human GPCR receptors were stimulated, and Ca2+ changes in numerous single cells were recorded over time using a fluorescent microscope and a CCD camera. We determined the percentage of live cells in a population responding to stimuli, the distribution of responses within a population of clonal cells, and the number of outliers. By employing principal component analysis and K-nearest neighbor modeling, we classified the time-resolved Ca2+ traces of single cells as a function of the stimulus type with high certainty for a population of cells. This method is potentially a powerful tool for identifying new drug targets or for investigating the single-cell behavior of an existing target or known receptor. The development of this single-cell drug screening method is presented, and fluorescent and statistical analyses of single-cell dynamic responses are discussed.