Surface antigen detection with non-fluorescent, antibody-coated microbeads: An alternative method compatible with conventional fluorochrome-based labeling

Background: Our goal was to devise a new labeling technique allowing the fl ow cytometric detection of an additional cell surface marker without the ne ed for a supplementary fluorochrome. Methods: Non-fluorescent polystyrene latex microbeads (0.1 or 0.5 mu m in d iameter) were coated with monoclonal antibodies (mAbs) by adsorption. Upon binding to their specific antigen on the surface of the cell, mAb-coated be ads induced a dramatic shift in the side scatter channel (SSC), resulting i n a well-defined cell population. Results: We show that expression of CD4 on murine peripheral lymphocytes, l abeled with anti-CD4 mAb-coated beads, can be readily detected through an a mplification of the SSC signal. Simultaneous labeling of lymphocytes with p hycoerythrin-(E)-conjugated anti-CD4 mAb and anti-CD4 mAb-coated beads, sho wed that all PE+ cells were SSChigh, thus establishing the specificity of t he technique. Hence, we have demonstrated that staining with mAb-coated bea ds could be combined to conventional labeling methods with fluoro chrome-co njugated mAbs. Using a standard 488 nm single laser cytometer, we have perf ormed a five-parameter analysis, simultaneously detecting fluorescein isoth iocyanate (FITC), PE, RED670(TM) and REDG13(TM), in combination with SSC si gnal modulation induced by mAb-coated beads. Moreover, are have shown that beads coated with mAbs directed against various antigens (CD45R, Mac-1, and TCR beta) can be used on various tissues, namely lymph nodes, spleen, or b one marrow. Conclusions: mAb-coated latex beads can therefore easily be used as an addi tional surface label, and provide a simple and reliable mean to upgrade the analysis capabilities of standard flow cytometry units. Cytometry 36:27-35 , 1999. (C) 1999 Wiley-Liss, Inc.