Multi-parameter flow cytometric analysis of uterine immune cell fluctuations over the murine estrous cycle

- A protocol for making single cell suspensions from mouse uterine tissue is described.
- Antibody panels for multi-parameter flow cytometric analysis of immune cells are listed.
- Macrophages and eosinophils are both identified by anti-F4/80 and CD11b antibodies.
- Additional use of SiglecF or FSC/SSC dotplots is suggested to separate these cell types.
- Most immune cell populations peak at estrus and metestrus phases of the estrous cycle.

Investigating immune cell populations within various reproductive tissues commonly utilises flow cytometric methods. With advances in fluorophore technology and equipment capabilities, multiple cell types from a single tissue sample can be identified by using different combinations of cell surface markers to distinguish specific cell populations. Here a protocol optimized for mouse uterine tissue was used to show the proportional changes in dendritic cells, monocyte/macrophages, T and B cells, NK and NK T cells, and the granulocytes, neutrophils and eosinophils at each of the four stages of the estrous cycle. Importantly, we demonstrate that use of anti-SiglecF or assessment of FSC/SSC plots could be used to differentiate monocyte/macrophage and eosinophil populations that otherwise cannot be distinguished by use of the common combination of antibodies against F4/80 and CD11b. Our results clearly indicate that within the uterus a dynamic population of immune cells resides, with many cell types reaching peak abundance at estrus and metestrus phases of the cycle, consistent with their importance in the response to paternal antigens and/or pathogens encountered after insemination.