Hi Yoav, we have used Becton Dickinsionīs TruCount beads (defined number of beads in FACS tubes, see http://www.bdbiosciences.com/immunocytometry_systems/) with good results and calculated absolute cell numbers from total events (e.g. 10,000 to 15,000) using cell and bead counts within the mixture after gating on beads and PI (propidium iodide) negative live and/or PI positive dead cells mainly according to BDīs protocol. However, for an accurate calculation proper mixtures/proportions of beads and cells (not to many cells!) are necessary. The major disadvantage is that - as far as I know - BD does not offer the beads separately under normal conditions. Nevertheless I would ask ..... Best regards Georg Kreysch yaltman@liai.org am 15.03.2002 23:52:01 An: cytometry@flowcyt.cyto.purdue.edu Kopie: Thema: consistency in counting cells by flow 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
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