For bacteria I use ratiometric bead counting and flow cytometry since the mid eighties very successfully. For mammalian cells we tend to use the absolute count facility in the EPICS XL, normally 60 to 100ul (3 or 5* 20ul cycle). The counting variability actually just done for yeast cells yesterday followed nicely the statistical counting error (n)^0.5/n in %, e. g 1.46% for 6153 cells counted (1.29% theoretical for 6000 counts) and 0.74% for 23585 (0.65% theoretical) for ten sample replicates. Problems come from cells clumping, sticking to plastic surfaces or sedimenting which also can be a problem for ratiometric counting. Additional problems come with system dead times if you use this method. When you use bead counting ensure you check for static loss and in particular for bead / cell coincidence of aggregates. It is also not working well if some of your cells eat your beads. Also consider that the statistical variation of the ratio is the sum of the two variations. We actually make our own bead standards and count them out using the EPICS XL as a very sophisticated 'Coulter counter' Regards Gerhard -----Original Message----- From: Karim Vermaelen [SMTP:Karim.Vermaelen@rug.ac.be] Sent: Thursday, June 14, 2001 4:32 PM To: Cytometry Mailing List Subject: absolute cell counts using FACS x-mac-creator="4D4F5353" Content-Transfer-Encoding: 7bit Hi everybody , Can anyone recommend a microsphere kit for performing simple cell counts on numerous samples using the FACS? BD's Truecount beads are only sold along with a MAb. Our cells do not have to be labelled. Any simpe alternative? (NB: our good ol' Coulter particle counter is dead) Tx Karim << File: Card for Karim Vermaelen >>
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