Rare-event sorting by fixed-time flow cytometry based on changes in intracellular free calcium

Neurotransmitter induced intracellular free calcium, [Ca2+]i , increases were used to sort rare responding cells by fixed-time flow cytometry. Non-transfected and transfected NIH/3T3 mouse fibroblasts were stimulated with a cocktail of the neurotransmitters oxytocin, serotonin, substance P, noradrenalin, vasopressin, and neurotensin. In both cultures 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 cultivation, cell cultures with more than 60% serotonin responsive cells were obtained. The initial frequency of these responders was less than 3 x 10(-4). NIH/3T3 cells transfected with total genomic DNA from the rat pituitary-gland cell line GH3 were sorted with a cocktail without serotonin. After two sorts and subsequent cloning two clones were obtained. Each of these clones was sensitive to one component of the cocktail (oxytocin or substance P). The initial frequency of one responder type was estimated to be less than 2 x 10(-5). These results demonstrate that sorting by fixed-time flow cytometry is a sensitive tool to enrich very rare cells from heterogeneous cultures.