A 21-Color Spectral Flow Cytometry Panel for Comprehensive Immunophenotyping of Rat Tissues and a Companion 9-Color T-Cell Functional Assay

Abstract

Although rats are crucial translational research models due to their immunological homology and surgical feasibility, the scarcity of conjugated antibodies has hampered high-dimensional immune profiling. To bridge this gap, we created a synergistic two-panel method that uses full-spectral flow cytometry: a 21-color comprehensive immunophenotyping panel and a 9-color intracellular cytokine staining (ICS) panel. The 21-color panel navigates the limited fluorophore pool and maps innate and adaptive lineages at high resolution. While it can be used to profile neutrophils and monocytes in peripheral blood it reaches its full potential in lymphoid organs, allowing for the simultaneous evaluation of tissue-resident macrophages, dendritic cells, T cell differentiation (including Tregs), and APC function. Based on this phenotypic paradigm, the 9-color ICS panel dissects effector T cell activation and Th1/Th2/Th17 cytokine production. Furthermore, we reserved the PE color during panel creation, allowing for the use of antibodies and probes with PE or comparable fluorophores in conjunction with this panel, hence increasing overall flexibility. This combination technique, designed for rat peripheral blood, spleen, and lymph nodes, combines panoramic phenotyping with functional analysis, offering an adaptable foundation for complicated tissue microenvironments in tumor immunology, rheumatology, and autoimmunity research.