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They excited at 457 nm; I later tried 441 nm, which should have been better,
but didn't get good discrimination (I was measuring polarized and
depolarized scatter at the same time). However, after I tweaked up the
Cytomutt to improve fluorescence sensitivity, I was able to identify a
cluster of eosinophils by 488->520 nm fluorescence; when compared to Flow
Cytometry Standards' fluorescein beads, eosinophil fluorescence is about
5,000 MESF, whereas the rest of the leukocytes are 1,000 or fewer MESF. On
a modern instrument with high fluorescence sensitivity (Elite, FACSort,
FACSCalibur), or on an old Ortho (where the relay fiber optics will mess up
polarization), it may be easier to sort on autofluorescence than on
polarized scatter; on stream-in-air systems, scatter is probably a better bet.
--Howard Shapiro
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