Flow cytometric identification and purification of cells by ligand-induced changes in intracellular calcium

Abstract

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 [Ca 2+] is an important part of the cellular response to SP. In our studies, SP-induced elevation of [Ca 2+] 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 [Ca 2+] i by using a dual injector system for agonist and cells, and a γ connector for mixing. These techniques were developed initially using cell lines and have been applied to acutely dissociated rat spinal cord cells.