Flow Cytometric Identification and Purification of Cells by Ligand-induced Changes in Intracellular Calcium

We have developed a method for identification and purification of functional subpopulations of cells defined by selective agonist activation. In a variety of cell types expressing substance P (SP) receptors, an increase in [Ca2+]i is an important part of the cellular response to SP. In our studies, SP-induced elevation of [Ca2+]i was monitored with the calcium indicator indo-1 and detected using a fluorescence-sensitive flow cytometer. Since responses to SP are transient in the continued presence of the peptide, a method was developed to set a fixed, brief interval between exposure of cells to peptide and measurement of [Ca2+]i by using a dual injector system for agonist and cells, and a y connector for mixing. These techniques were developed initially using cell lines and have been applied to acutely dissociated rat spinal cord cells.