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In summary, a dilute solution of antibody was run as a sample on a BD bench at a
known flow rate and jet speed, producing a DC signal of whose amplitude could be
measured. Uniform microspheres run under the same conditions produced pulses
whose area (in terms of signal level X time) were measured. From these two
measurements we calculated how many reagent molecules would have to be bound to
a cell to produce a pulse equal to that of a standard microshpere. Signals from
cells stained with the same antibody were compared to the "calibrated"
microshpere signals to compute an "absolute" antigen density, and gave good
agreement with data obtained by the radiometric method. This method, however,
did not yield an absolute calibration standard that could be moved from machine
to machine, or compensate for daily variations on the same instrument.
Our methodology, of course, was dependent upon an antibody-fluorochrome
conjugate having the same fluorescence whether or not it is bound to a cell,
measurement conditions being constant, and no energy transfer ocurring. Does
anyone have any information as to whether this is true or false?
-Marty Bigos
Stanford Shared FACS Facility
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