Development of a dried pre-formulated antibody panel (7 colors/17antibodies) for the identification of human innate lymphoid cells by flow cytometry

Abstract

Abstract Innate lymphoid cells (ILC) play an important role in innate immunity, lymphoid organogenesis and tissue remodeling. They form a heterogeneous population that can be categorized into 3 groups (i.e. ILC1, ILC2 and ILC3) based on their phenotype and/or distinct patterns of cytokine production. The identification and enumeration of different blood ILC subsets is important to understand immune regulation in disease models such as allergy, cancer and inflammation. Flow Cytometry is the method of choice for analysis of ILCs in human blood. However, as ILC frequencies may range below 0.1% of white blood cells, considerably elevated sample volumes and/or cell-enrichment is required. Furthermore, due to the lack of specific markers for definition of ILCs, at least 15 antibodies are required for mere identification of ILCs, including a lineage-exclusion channel to exclude most of the leukocytes. Based on current knowledge in human ILC biology, we have composed a respective set of markers to design a dry unitized 17 antibodies/7-color panel (CD294/Lineage/CD117/Nkp46/CD127/CD161/CD45) for flow cytometry. The panel in dry DURAClone format allows for the reliable identification of all ILCs subsets in whole blood or peripheral blood mononuclear cell fractions (PBMCs) in concordance with current scientific opinion. Due to the elimination of the pipetting of 17 antibodies, the method can substantially reduce variability and error related to sample preparation. Furthermore, the streamlined workflow can facilitate inter-laboratory transfer of the assays in multi-site studies.