Three‐color immunofluorescence analysis of mouse B‐lymphocyte subpopulations

Abstract

We have modified a fluorescence‐activated cell sorter (FACS) to make three independent immunofluorescence measurements on each cell and used this system to study mouse B‐lymphocyte subpopulations. An argon‐ion laser (emitting at 488 nm) excites fluorescein‐and phycoerythrin‐labeled reagents, and a tunable dye laser charged with rhodamine 6G (emitting at 615 nm) excites an allophycocyanin‐labeled reagent. We report simultaneous measurements of IgM, IgD, and the recently‐defined mouse B lymphocyte antigens BLA‐1 and BLA‐2 on splenic lymphocytes of CBA/J mice and mice of the congenic strain CBA/N (which have an X‐linked immunodeficiency [xid]). These data provide information on relationships among the B‐cell populations in CBA/J “normal” mice and the defective CBA/N that could not be derived from one‐ or two‐ color immunofluorescent measurements. We believe this is the first use of allophycocyanin as an immunofluorescence label. Copyright © 1984 Wiley‐Liss, Inc.