absolute cell counts using FACS
donnenbergad at MSX.UPMC.EDU
Mon Jun 18 14:27:24 EST 2001
I agree with Mark that there is no magic to the single platform assay and
that many types of beads will do. We used to use glutaraldehyde-fixed
chicken red blood cells: broadly fluorescent, stable for years and
The trick that makes the clinical assays work so well is the lyse/no wash
protocol. As soon as a wash step is involved there is the danger of loss of
cells and/or beads with no assurance that loss is proportionate.
The lyse/no wash protocol leaves a lot of debris and requires that one of
the fluorescence channels be used for CD45 (if it is important to get a
"total leukocyte" denominator).
If you wish to get a simple nucleated cell count, PI staining of
permeabilized cells, plus the bead du jour will do the trick. One further
caveat: In the single platform assay, pipetting accuracy (of beads and
cells) is everything.
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)
-------------- next part --------------
HTML attachment scrubbed and removed
More information about the Cytometry