Pairs of violet-light-excited fluorochromes for flow cytometric analysis

We describe pairs of fluorochromes for use with the 407-nm line of a violet-light-enhanced krypton ion laser. These fluorochromes and a previously described violet-light-excited reporter variant, GFP-Vex, fall into two emission classes: blue for Cascade Blue, and green/yellow for Cascade Yellow, Lucifer Yellow, and GFP-Vex. Cascade Yellow is a new fluorochrome that we have synthesized and is used for the first time in the present study. The two emission classes are sufficiently different that Cascade Blue can be paired with Cascade Yellow, Lucifer Yellow, or GFP-Vex in flow cytometric analysis. Furthermore, with proper detection filters, these fluorochromes can be combined with all of the currently used fluorochromes in a three-laser FACS system. With these data, the total number of fluorochromes that can be used as antibody labels for simultaneous detection in combined FACS analysis increases to nine. This study demonstrates the sensitivity and power of the combined use of these reagents in a single eight-color analysis by identifying murine T-lymphocyte subsets that could not otherwise be readily distinguished.