Novel monocyte sub-populations in Sprague Dawley rats revealed by detailed analysis of CD43 and CD11b expression using a single-stain 11-colour fluorescence activated cell sorting protocol

Abstract

Abstract Monocytes are a diverse population of white blood cells with critical roles in immunity and inflammation. The rat is commonly used for immunological investigations, but comprehensive characterization of rat monocytes/macrophages has been hampered by limited accessibility to immunophenotyping capabilities in hospitals and medical research institutions. Here, we developed a single-stain 11-color fluorescence activated cell sorting protocol to identify and enumerate major peripheral blood mononuclear cell subgroups (pan T cells, helper T cells, cytotoxic T cells, NK cells, B cells) and monocyte sub-populations in healthy Sprague-Dawley rats. Cell surface marker expression and frequency of populations was validated in 8 male and 13 female rats and compared between young vs aged and cryopreserved vs fresh samples. We identified four putative monocyte sub-populations based on differential CD43 and CD11b expression, including two novel sub-populations (CD43Lo/CD11bLo and CD43Hi/CD11bLo) not previously described in rats. Each sub-population exhibited distinct cell morphology and surface marker profiles. Monocyte sub-population frequencies and surface expression were robust across sex, age and cryopreservation status. Immunophenotypically distinct monocyte sub-populations are increasingly recognized in both humans and mice, but only two sub-populations (CD43Hi and CD43Lo) have been described thus far for rats. Our data underscore the complexity and species-dependent variability of monocyte sub-populations and highlight the presence of at least four distinct monocyte sub-populations in Sprague Dawley rats. This validated panel will support consistency in rat immunological research, warranting further in-depth functional characterization of novel monocyte sub-populations.