From: Marcus Reckermann (recker@ftz-west.uni-kiel.de)
Date: Wed Feb 25 1998 - 07:57:08 EST
> Subject: absolute cell counting by flow. > Date: Mon, 23 Feb 1998 23:38:58 -0000 > From: Aki Hoji <akhst7+@pitt.edu> > To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> > > Dear, > > I am looking for a way to accurately count absolute cell numbers (from a > limited number of cells) by flow. I'd appreciate if anyone could send me > a working protocol to do this, or provide some refs. Thanks in advance. > Dear Aki, well, depending on the instrument you use, you have different options. As far as I know, only instruments from Partec (and possibly BioRad) have hardware options for absolute volume counts. The mostly used BD and Coulter instruments donīt have that (as far as I know), and you have to resort to indirect methods: - you can add a known bead concentration, and then calculate the event number of your cluster relative to the known bead concentration in your sample. BD distributes so-called TruCount Beads that facilitate this procedure. - if you have a reliably stable sample flow (check that thoroughly!), you can relate the elapsed time with the measured volume. - I use another trick: I fill the fluidics system with the sample to be measured, then stop the sample flow. Then I fill a tube with the exact volume I want to measure, and mount that onto the sample holder. I start the measurement simultaneousely on the instrument and on the aquisition software; I stop it simultaneousely when the entire volume has been pushed out and I can see air entering the exit tubing (which must reach down to the very bottom of the sample tube). This only works when you have a transparent sample and exit tubing. There is no dead volume in the sample line because I fill the system with the sample before I start measuring. - another fairly simple method is to weight your sample tube before and after your measurement. You just have to calibrate a sample volume/weight ratio for each sample type. I am curious to learn more ways of doing absolute volume counts! Good luck, marcus __________________________________________________ Dr. Marcus Reckermann Research- and Technology Centre Westcoast (FTZ) Hafentoern D-25761 Buesum Germany Tel: +49-4834-604-204 or -261 Fax: +49-4834-604-299 E-Mail: recker@ftz-west.uni-kiel.de
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