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

Abstract

Background: Our goal was to devise a new labeling technique allowing the flow cytometric detection of an additional cell surface marker without the need for a supplementary fluorochrome.

Methods: Non-fluorescent polystyrene latex microbeads (0.1 or 0.5 microm in diameter) were coated with monoclonal antibodies (mAbs) by adsorption. Upon binding to their specific antigen on the surface of the cell, mAb-coated beads induced a dramatic shift in the side scatter channel (SSC), resulting in a well-defined cell population.

Results: We show that expression of CD4 on murine peripheral lymphocytes, labeled with anti-CD4 mAb-coated beads, can be readily detected through an amplification of the SSC signal. Simultaneous labeling of lymphocytes with phycoerythrin- (PE)-conjugated anti-CD4 mAb and anti-CD4 mAb-coated beads, showed that all PE+ cells were SSChigh, thus establishing the specificity of the technique. Hence, we have demonstrated that staining with mAb-coated beads could be combined to conventional labeling methods with fluorochrome-conjugated mAbs. Using a standard 488 nm single laser cytometer, we have performed a five-parameter analysis, simultaneously detecting fluorescein isothiocyanate (FITC), PE, RED670 and RED613, in combination with SSC signal modulation induced by mAb-coated beads. Moreover, we have shown that beads coated with mAbs directed against various antigens (CD45R, Mac-1, and TCRbeta) can be used on various tissues, namely lymph nodes, spleen, or bone marrow.

Conclusions: mAb-coated latex beads can therefore easily be used as an additional surface label, and provide a simple and reliable mean to upgrade the analysis capabilities of standard flow cytometry units.