The excitation spectrum and emission spectra (bold line) are shown for
the tandem dye Cy5PE. The red-shaded region represents the bandpass filter
commonly used to measure Cy5PE fluorescence. On most flow cytometers, Cy5PE
is excited by an argon laser (tuned to 488 nm). Depending on the degree
of quenching of PE fluorescence by the Cy5, different degrees of compensation
will be required with the PE channel (excitation at 575 nm). Cy5 is also
very well excited by the 633 nm line of a HeNe laser, make the Cy5PE dye
difficult to use in conjunction with APC. However, when using a dye laser
(595-605 nm) to detect APC, the problem is considerably less, since Cy5
is not efficiently excited by those wavelengths. Intralaser compensation
is required whenever this dye is used simultaneously with APC.
Compare the Cy5PE fluorescence spectra to spectra for several FACS immunofluorescence dyes.