Having modified a FACStar+ cell sorter to cope with eight detected parameters including six fluorescence signals excited by illumination at 351-364nm (UV), 488nm and 605nm in two beams, attention turns to selecting a suitable set of immunofluorochromes from the presently available collection.
Four fluorochromes routinely used in combination are Fluorescein (FITC), Phycoerythrin (PE), Texas Red (Tex) and Allophycocyanine (APC). We have investigated the possibility of adding to these either PerCP or Tricolor, each of which is excited at 488 nm and emits in the deep red, as the 5th fluorescence colour and Cascade Blue, excited in the UV and emitting in the blue region, as the 6th colour. In seeking the optimal combination of laser powers, optical filters and detector types and sensitivities, the criteria tested are the brightness of each fluorescence signal relative to background levels and the degree of spillover of each emission into the detectors of its peers and vice versa. This spillover is a particular problem in those sorters which cannot perform fluorescence compensation between signals generated at two time points (by displaced laser beams).
Thus, it has been determined, for example, that low level leakage of FITC emissions into the Cascade Blue channel and vice versa, was unavoidable but tolerable, that, while PerCP is at a large intensity disadvantage relative to Tricolor as the 5th colour, its leakage into the APC channel is lower and that, since PerCP is detected through a very long wavelength (680DF23) optical filter, no significant leakage of other fluorochromes into that channel occurs.