Multi-parameter flow sorting with novel antibody fluorochrome conjugates that facilitate relabeling of targeted epitopes and reuse of fluorescence channels in downstream applications

Abstract

Abstract Multi-parameter immunofluorescent labeling is the method of choice for flow sorting of cell populations out of heterogeneous mixtures. However, downstream applications of isolated cells are limited since fluorescence channels and epitopes are blocked by antibody fluorochrome conjugates utilized for the flow sorting experiment. Here, we describe for the first time new types of antibody fluorochrome conjugates that enable a highly specific multi-parameter cell labeling. Unlike conventional fluorochrome-conjugated antibodies, the introduced conjugates rely on recombinantly engineered antibodies and a conjugation chemistry that facilitates the release of the whole conjugates from the cell surface after the flow sorting step. Thereby, previously blocked epitopes are free for relabeling, which provides maximal flexibility for renewed epitope targeting in downstream applications. We demonstrate this flexibility in the context of a workflow for the isolation of highly pure human regulatory T cells (Treg). Tregs labeled with conventional CD4-VioBlue, CD25-PE and the new releasable CD127-APC were enriched by flow sorting. Final identification of the target cells was achieved by intranuclear labeling of the transcription factor FoxP3, a low expressed marker which requires a bright fluorochrome and fluorescence channel with high sensitivity for reliable detection. The selective removal of the CD127-APC conjugate facilitated the reuse of the APC-channel and enables maximum sensitivity for the cell sorting and analysis step. In summary, this new generation of releasable antibody fluorochrome conjugates for multi-parameter cell labeling paves the way for so far impossible workflows in research and clinical applications.