This is a problem that has no single solution. In my experience I have lost cells due to staining/washing, lysis, adherence to various tube materials, phases of the moon, etc. There have been any number of individuals (myself included) who have tried to make an explantation to some irate individual who put 10,000 cells into 1 ml and wanted to know why I was not able to give them 10,000 events (sometimes they wanted 10,000 sorted cells, but that is another story). One approach to take is to get some large (cell-sized) fluorescent beads, put 10,000 of them into the volume you normally use, and run them through the instrument. The advantage there is that you have consistent density & fluorescence that you can trigger on and eliminate the "no-see-ums" that seem to be inherent in any biological population. This should give you a handle on what % of the population you are losing due to instrumental anomalies and set a baseline from which to start looking for the disappearing cells (you can work with any number of beads, as long as you know what it is, and calculate your % loss from that). Keith ********************************************************************** * "If you get to Keith A. Kelley kelley@wh.bayer.com * thinking you're a Institute for Research Technologies * person of some Bayer Corporation * influence, try 400 Morgan Lane Ph 203-931-5303 * ordering someone West Haven CT 06516 FAX 203-937-5467 * else's dog around" * Texas Bix Bender **********************************************************************
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