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.
Schieren
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Howard Hughes Medical Institute, College of Physicians and Surgeons, Columbia University, New York, NY 10032.
MacDermott
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D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods*; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter / Q000187:drug effects / Q000378:metabolism*; D013116:Spinal Cord / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D013373:Substance P / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism
D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter; D013373:Substance P; D002118:Calcium