56
were placed on the platelets and the beads, and fluorescence histograms generated. In
addition, unconjugated beads of the same type were also added. The difference in fluorescence
IS;
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Figure 3. A dot plot showing forward scatter versus side scatter illustrates a mixture of platelets and beads.
Gating on the platelets yields a FLl histogram with a broad distribution. Gating on the ead singlets yields
an FLI histogram with two peaks corresponding to unlabelled beads and beads containing a known amount
of antigen (see text).
levels between the unconjugated beads and the labeled beads was used to calibrate the
fluorescence scale; the amount of IgG per platelet could be calculated from the mean
fluorescence of the platelets.
This procedure involves a number of assumptions. First, that the beads and platelets are
labeled in the same way in the mixture as they would be if labeled separately. This is easily
tested, and proved to be the case in this example (Figure 4).