RARE-EVENT SORTING BY FIXED-TIME FLOW-CYTOMETRY BASED ON CHANGES IN INTRACELLULAR FREE CALCIUM

Neurotransmitter induced intracellular free calcium, [Ca2+](i), increa ses were used to sort rare responding cells by fixed-time now cytometr y, Non-transfected and transfected NIH/3T3 mouse fibroblasts were stim ulated with a cocktail of the neurotransmitters oxytocin, serotonin, s ubstance P, noradrenalin, vasopressin, and neurotensin. In both cultur es no detectable response to this cocktail was found. Non-transfected cells were stimulated with the cocktail and sorted for rare responders , After two subsequent aseptic sorts, each followed by subsequent cult ivation, cell cultures with more than 60% serotonin responsive cells w ere obtained. The initial frequency of these responders was less than 3x10(-4), NIH/3T3 cells transfected with total genomic DNA from the ra t pituitary-gland cell line GH3 were sorted with a cocktail without se rotonin. After two sorts and subsequent cloning two clones were obtain ed. Each of these clones was sensitive to one component of the cocktai l (oxytocin or substance P). The initial frequency of one responder ty pe was estimated to be less than 2x10(-5). These results demonstrate t hat sorting by fixed-time now cytometry is a sensitive tool to enrich very rare cells from heterogeneous cultures. (C) 1997 Wiley-Liss, Inc.