I have a colleague who is thinking about using flow cytometry to screen a population of cells with a fluorescently labeled peptide ligand. The problem is that an extremely low number of these cells will be expressing the receptor for the ligand... probably less than one in 25,000. The cells will be otherwise identical, so we can't use any other markers for subset analysis. (Although we are considering ways to use GFP... I'm not really clear on the molecular biology of this system.) My question: if flow cytometry an appropriate tool for this assay? Can you think of a better tool? If we do use flow, what fluorophore should we conjugate to the ligand? I would like to use something really bright, so that I can resolve the peaks even at a high flow rate. I would also like to use something small, to minimize the chances or the fluorophore causing steric hindrance. The Kd for the ligand is around 5 nM... should we wash the sample after incubating with the ligand? Thanks in advance for any suggestions. Mark A. Miller Pharmacology Cell Sorting Lab WP27E-120 Mailbox WP26-265 Phone (215) 652 4597
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