Cytometry of Rare Surface Molecules by Magnetofluorescent Liposomes

Abstract

The detection limit of conventional immunofluorescence is in the range of several thousand molecules per cell, depending on the cellular autofluorescence and type and quality of reagents [1]. Many molecules are expressed in lower copy numbers, like receptors for cytokines, hormones or growth factors, and are difficult to analyse [2]. Nevertheless, these molecules are often of high functional relevance, defining cellular subsets with specialized functions or differentiation status. One major obstacle of conventional fluorescence-labelled antibodies is that only 1–10 dye molecules can be conjugated to a single antibody molecule. Magnetofluorescent liposomes, containing magnetic particles and thousands of fluorescein molecules and conjugated to specific antibodies can overcome this problem. Compared to conventional reagents they increase fluorescence signal intensity up to 1000-fold [3]. Magnetofluorescent liposomes allow unambiguous cytometric detection of cells according to expression of less than 300–400 antigens per cell. In addition, they offer the option of isolating labelled cells by MACS or FACS. So far, liposomes have been used to analyse the expression of CD25 on a subpopulation of resting B- and T cells [3] and to investigate the expression of IL-6- and IL-3 receptors on CD34⁺ hematopoietic stem cells. They also allowed the demonstration of the specific expression of IFN-γ and IL-10 in low abundancy on the surface of cells secreting these cytokines [4, 5].