Isolation of mononuclear phagocytes from the mouse gut

The intestinal tract is home to trillions of microbes that make up the gut microbiota and is a major source of environmental antigens that can be derived from food, commensal microorganisms, and potential pathogens. Amidst this complex environment, myeloid cells, including macrophages (MPs) and dendritic cells (DCs), are key immunological sentinels that locally maintain both tissue and immune homeostasis. Recent research has revealed substantial functional and developmental heterogeneity within the intestinal DC and MP compartments, with evidence pointing to their regulation by the microbiota. DCs are classically divided into three subsets based on their CD103 and CD11b expression: CD103⁺CD11b^-(XCR1⁺) cDC1s, CD103⁺CD11b⁺ cDC2s, and CD103^-CD11b⁺ cDC2s. Meanwhile, mature gut MPs have recently been classified by their expression of Tim-4 and CD4 into a long-lived, self-maintaining Tim-4⁺CD4⁺ population and short-lived, monocyte-derived Tim-4^-CD4⁺ and Tim-4^-CD4^- populations. In this chapter, we provide experimental procedures to classify and isolate these myeloid subsets from the murine intestinal lamina propria for functional characterization.