After so much discussion about how to do absolute FCM counts with beads, here
is my complete procedure, which works for me since 20 (!) years.
1. Find a type of bead that sits well in all your histograms/dotplots, away
from any important cell cluster location.
My favourites, nowadays, are Coulter Immuno-Brite Level IV. They make sharp
peaks in all channels, also the UV-excited fluorescence. That makes them
useful for alignment checks, too. I use old leftover counting samples to do
all my alignments.
2. Mix a vial of those beads (after some sonication and vortexing) with 100
ml of PBS. That gives a useful density for most FCM cell countings.
If you are using cell dyes that leak upon dilution, make the beads up in that
dye solution.
3. Aliquot 0.5 ml portions into empty FCM sample tubes. Use repeat pipettor
and keep suspension well suspended during the whole act.
Obviously, the 100 ml and 0.5 ml are not essential, it's just what I do.
4. Put tubes into freezer (!)
5. When you get around to it, take a few tubes, thaw them up, vortex, and
count the beads on the Coulter counter. Calculate total number of beads per
tube.
If you are not sure with your beads about thresholds etc on your Coulter
counter, count cells instead, mix them with beads, run the mixture on your
FCM, and get your absolute bead numbers this way around.
6. Whenever you have a FCM-prepared cell sample you want to count, thaw up a
bead tube, vortex, add the cell suspension, run on the FCM right away.
Don't let the cell/bead suspension stand around for too long. Those beads
have a way of hiding if they are given the time.
7. Determine ratio of cells / beads from any of the histograms/dotplots, and
calculate total numbers of cells in your tube from the known total number of
beads.
The cell sample volumes added into the tubes are irrelevant for the
calculation (!)
Good luck
Ralph M. Bohmer
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