On Fri, 29 Oct 1999, Snezna Rogelj wrote: > I do not have a Coulter counter, and am cross with the hemacytometer. > Can I use the FACScan to count cells in a defined volume, > i.e. to calculate cell concentrations? Hi Snezna, I have had a few requests in the past to turn the FACScan into a rather high tech haemocytometer but now I have a few users who are using it to assess 'absolute numbers' of cells in successive generations judged by CFSE staining. The way we are doing this is to spike the sample with a small aliquot of beads. If you have a solution of beads at a known concentration (this is assessed by haemocytometer so you have to use it once I am afraid - dont be cross with it!), add a known volume, run your sample and calculate 'absolute cell count' per µl by: (No. of cells in defined region/No. of beads in bead region)*(Total no. of beads/sample vol in µl) I say 'absolute' as there will be errors especially those introduced by pipetting errors but it does seem to work surprisingly well - I suppose you could use something like BD's TruCount as well. I use Calibrite beads as they are a reasonable size but it may be necessary to use a log scale for forward scatter to get both the bead and the cell population visible. It also has the advantage over haemocytometer in that you can get counts on phenotypically defined cells. A literature search on absolute cell count will yield a number of references. Good luck, Derek ************************************************************************ Derek Davies Voice: (44) 0171 269 3394 FACS Laboratory, FAX: (44) 0171 269 3100 Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk London, UK Web Page: http://www.icnet.uk/axp/facs/davies/index.html In tenebris lux *************************************************************************
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