Detection of triple antibody-binding lymphocytes in standard single laser flow cytometry using colloidal gold, fluorescein and phycoerythrin as labels

Abstract

Goat anti-mouse antibodies conjugated with colloidal 40 nm gold particles (G40) were used as secondary layers to stain human T lymphocytes in an attempt to extend the polarized light epi-illumination microscopic technique to flow cytometric multiparameter analysis. G40-labelled T cells were further stained with phycoerythrin (PE, red)- and fluorescein (FITC, green)-conjugated antibodies with specificity for the same cells. The cell samples were then analysed on a standard flow cytometer equipped with one laser operating at 488 nm. G40-labelled cells were detected in the side scatter (90°) channel and fluorescent cells in the red and green fluorescence channels, respectively. Single labelling of the same T cell subsets with either G40-, FITC- or PE-conjugated antibodies yielded similar results, and cell mixture experiments did not show interference between gold and fluorescence labels. Triple staining experiments with three differently conjugated antibodies showed that subpopulations of cells were labelled in an independent manner with one, two or three antibodies in proportions expected from fluorescence controls. It was, therefore, possible to detect subset, sub-subset and activation markers on individual T cells. Our experiments show that immunogold staining of cell populations can be detected in routine one-laser flow cytometry. Further, the gold label can be combined to give two-color staining with ordinary red and green fluorochrome-conjugated antibodies thereby permitting rapid and precise triple antibody staining of individual cells. This methodology provides a powerful tool for the analysis of cellular antigenic diversity among, e.g., immunocompetent cells.