Mono-Chlorinated 3-Carboxy-7-Hydroxycoumarin: A Highly Fluorescent and Water-Soluble Violet Excitable Dye for Cell Analysis

Abstract In our search for new violet excitable dyes with improved photochemical properties, we examined several halogen substituted 7-hydroxycoumarins and found that chlorinated derivatives could be as bright as their fluorinated analogs. In particular, mono-chlorinated 3-carboxy-7-hydroxycoumarin (V450) has a quantum yield equal to 3-carboxy-6,8- difluoro-7-hydroxycoumarin (Pacific Blue™). Antibodies specific to various cell surface markers (CD3, CD4, CD45) were labeled with V450 and tested in single color flow cytometry assays. Our results show that conjugates of V450 are brighter than the same clones labeled with Pacific Blue™. Additional studies were performed in which V450-antibody conjugates were included in 8-color cocktails, along with FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, and APC-H7 conjugates, aimed at subsetting lymphocytes and myeloid cells. These cocktails met all standards of performance for the particular applications for which they were designed, including stability, lack of interaction between components, and brightness. The new V450 fluorochrome proved to be compatible with both FACSLyse and PharmLyse processing of EDTA anti-coagulated whole blood, and showed no signs of adverse effects on unfixed cells.