The new violet laser dye, Krome Orange, allows an optimal polychromatic immunophenotyping based on CD45-KO gating

• Published in: Journal of immunological methods 2011-09, Vol.372 (1), p.42-51
• Main Authors: Preijers, Frank W.M.B., Huys, Erik, Leenders, Marij, Nieto, Laura, Gautherot, Emmanuel, Moshaver, Bijan
• Format: Article
• Language: English
• Subjects: 10-color flow cytometry; Biological and medical sciences; CD45; cell differentiation; dyes; Flow Cytometry - instrumentation; Flow Cytometry - methods; fluorescence; Fluorescent Dyes - chemistry; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Humans; Immunophenotyping - methods; Krome Orange; Leukocyte Common Antigens - immunology; leukocytes; Leukocytes, Mononuclear - immunology; Molecular immunology; monoclonal antibodies; Small cell samples; Statistics, Nonparametric; Techniques; Violet-laser dye
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2011.06.025

Polychromatic immunophenotyping improves characterization of leukocyte subpopulations and their malignant counterparts. However, the lack of various fluorochrome-labeled monoclonal antibodies (MoAbs) hinders the formation of multi-color panels. CD45 appears to be an important MoAb for immunophenotyping of these cells. Plotted against the side scatter, CD45 provides immunological cell differentiation and the ability to recognize various normal and malignant leukocyte subpopulations. CD45 is commonly used and labeled with various fluorochromes and as a result, is incorporated in multi-color panels as a conjugate of less available fluorochromes, such as the violet laser dyes. However, these dyes (e.g. Pacific Orange/PO) often possess low fluorescence intensity, which may be too weak to differentiate between populations. The new organic dye Krome Orange (KO, emission at 528 nm) appears to be a more intense violet laser dye, serving as an alternative to PO. Intensities of CD45 conjugated with FITC, PE, ECD, PE-Cy5, PE-Cy7, PO and KO were tested in different cell sources. Various lineage markers were sequentially back gated on CD45-KO to identify subpopulations. A 10-color MoAb panel for determination of aberrancies in small cell samples was composed to test specificity of CD45-KO. We showed in various fixed and unfixed cells from different sources that KO is a suitable fluorochrome with a significantly higher quantum yield than PO and is even brighter than other violet laser dyes (e.g. Pacific Blue). CD45-KO/SS enables us to distinguish and characterize various normal and malignant leukocyte subpopulations. By using a 10-color MoAb panel to screen on aberrancies, we showed that CD45-KO provides reliable immunophenotyping within small amounts of cells and thereby improves the quality of 10-color stainings. ► The violet laser dye, Krome Orange TM, expresses higher quantum yield than Pacific Orange, even higher than other dyes. ► CD45-Krome Orange (CD45-KO) is suitable to be applied in polychromatic immunophenotyping. ► CD45-KO, combined with side scatter (SS), can be used to perform immunological differentiation. ► CD45-KO/SS enables to distinguish and characterize various normal and malignant leukocyte subpopulations. ► Using a 10-color MoAb panel to screen on aberrancies, CD45-KO provides reliable phenotyping of small cell samples.