absolute cell counts using FACS

Donnenberg, Albert donnenbergad at MSX.UPMC.EDU
Mon Jun 18 14:27:24 EST 2001


Karim-

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
essentially free.

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.

Albert


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

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