Simple counts with flow
Howard Shapiro
hms at shapirolab.com
Thu Feb 27 23:16:32 EST 1997
>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.
>
The problem with this approach is that the precision of the count of beads
per unit volume is determined by the sampling statistics of how many beads
you actually count in the hemacytometer; when you count n beads, the
standard deviation due to sampling error alone (i.e., in the absence of
dilution errors, etc.) is the square root of n. You'd therefore need to
count 10,000 beads on the hemacytometer to get a 1% CV in the number of
beads per unit volume. If you counted 1,000 beads, which is more than
people generally count by eye, sampling error alone would keep the CV at or
above 3.2%. If you counted 400 beads, the CV could be no better than 5%,
and so on. That's why the Coulter counter became popular for cell counting
within a few years of its introduction; it's a reasonably simple matter to
count 10,000 cells with such an instrument. If you have access to a Coulter
counter or other hematology instrument capable of absolute counting, you can
use that to get a more accurate and precise count of beads per unit volume
in your bead solution, and then aliquot beads into your samples to provide
an index for absolute counts of cells. Again, it will be necessary to count
thousands of the beads (and the cells of interest) to achieve high
precision. Ideally, one could maintain an arbitrary level of precision in
absolute counting by counting some fixed number of cells of interest in
every sample, and determining the volume of sample required to attain that
cell count.
-Howard
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