Simple counts with flow

Chris Groves chris_groves at
Thu Feb 27 08:32:36 EST 1997

Just my two cents worth...This is a lazy FACS man's method for cell counts by flow.

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 
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 = ratio of the number (percentage) of cells/beads as measured by a flow cytometer.

number of cells= 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!

Chris Groves
Millennium Pharmaceuticals Inc.
Cambridge, MA
groves at

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