FITC spectrum has significant overlap into the FL2 channel on Facscan. Moreover,
the spectrum of FITC in the FL3 channel is not the same as that of PE. So the
signal that results in the FL2 from FITC bright objects requires different
compensation than that of PE in the FL3 channel. Given the stock filters chosen
for the Facscan (585BP for FL2 and 650LP for FL3) the compensation of PE into
FL3 will be high enough to overcompensate FITC in FL3. Since there is no
compensator between FL1 and FL3 (that would go negative as well) there are no
controls that can be adjusted to correct this.
However, if the Facscan in question is not used for PI DNA analysis on FL3, a
partial solution would be to replace the FL3 filter (a 650 LP) with one better
optimized for the Cy5-PE spectrum. On our Facstar we use a 685 BP filter (which
is centered around the Cy5-PE peak). This would minimizes the FL2 to FL3
compensation thus reducing the FITC overcompensation in FL3. This filter also
allows enough PI signal to get though if it is used for live/dead cell gating;
but not enough for good quality DNA analysis.
-Marty Bigos & the gang
Stanford Shared FACS Facility