Make a concentrated solution of any brand of polystyrene microspheres in
ddH20, or saline,PBS, etc... The beads should be either twice or half the =
size of the cells of interest, so that the bead population should be =
easily identifiable. The important thing to note is that you want to be =
able to view both populations separately on an FSC vs. SSC cytogram.
Determine the concentration of the solution by counting the beads on an
hemocytometer. Do this at least 4x to make sure you have an accurate
representation.
Add a small volume (ie.100ul) of beads to a cell suspension of unknown
concentration, but known volume (ie. 900ul). Measure on a flow cytometer. =
Measure the total viable cell/bead population and count 10,000 events.
The side scatter (SSC, SS, RALS, Log90) should be measured in Log scale =
due to the difference in cell versus bead size. Depends on the relative =
sizes (um) of the cell population and the beads.
The concentration of cells per tube is calculated by using the =
relationship:
The ratio of the number of cells/beads =3D ratio of the number =
(percentage) of cells/beads as measured by a flow cytometer.
therefore:
number of cells=3D percentage of cells/beads as measured by flow =
cytometry
multiplied by the total number of beads added.
Now the concentration by volume may be calculated by dividing the cell
number by the initial volume of cells/beads.
This protocol was adapted from the method published by Sabine BlaB and
Klaus Lenmartz, Zeitschrift fur Naturforschung, 46c, 1130-1133. 1991.
Many vendors sell fluorescent microspheres.I use 9um beads for 4-6um =
cells. Shake those babies up (and/or ultrasonicate) to get an even =
suspension of beads before future aliquots into cells.
Happy counting!
Cheers,
Chris Groves
Millennium Pharmaceuticals Inc.
Cambridge, MA
groves@mpi.com
CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web