Hi Yoav I'm using Exalpha Perfectcount microspheres for enumeration. These contain two populations of beads with different sizes and fluorescences; 50% low and 50 % high fluorescence in all channels. The point is that unless the beads are properly mixed, you won't get a 50:50 mix. The only problem is if you run a sample for a long time the brighter (and larger) beads tend to settle.They need a really good mix immediately before use. The concentration is determined by the manufacturers although I tend to confirm this by manual counting. You may not need anything as fancy as this and there are a number of other products available. Good luck Simon Watson On 15 Mar 02, at 14:52, Yoav Altman wrote: > > Dear Flowers, > > A colleague of mine wishes to use a FACScan to count cells for an > experiment. The experiment will proceed over several weeks, and he > wishes to count live and dead cells over several time points (eg. 1 > wk, 2 wks, etc.) > > Assuming that his samples are resuspended in the same volume every > time, and that the flow rate would be the same from week to week or > sample to sample, he could just take the number of events in his live > gate vs. non-debris events. But, he is concerned that flow rate may > vary from week to week due to small obstructions in the fluidics. > > It seems that if we had a bead of known concentration we could add > these to the sample and do something like stop collecting after > 10,000 beads and thus know we had the same volume as last time. Does > this seem like a viable approach. Does anyone make beads in known > concentration, or is there some other standard we can use to know > that we are counting cells in the same volume from week to week. > > Thanks, > > Yoav > --- > Yoav Altman > Imaging Facility Specialist > La Jolla Institute for Allergy & Immunology > 10355 Science Center Drive > San Diego, CA 92121 > > email: <yaltman@liai.org> > Phone: (858) 678-4608 > Fax: (858) 558-3526 Dr Simon Watson MRC Clinical Research Fellow Phagocyte Lab First Floor Medical School Teviot Place Edinburgh EH10 4EB UK Tel: +44 (0)131 651 1570
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