Circulating TEMs are bona fide hematopoietic cells distinct from circulating ECs and express a resident monocyte phenotype. (A) Analysis of mouse liver by IFS (left) and flow cytometry (right). (Left panels) Expression of GFP (green) in Cd31⁺ (red) liver sinusoids of Tie2-GFPmir142T mice (n = 2). Scale bar represents 60 μm. Dot plots on the right show liver tissue–derived single-cell suspensions of representative wild-type (n = 1), Tie2-GFP (n = 2), and Tie2-GFPmir142T (n = 3) mice. Note that GFP is expressed robustly in the Cd31⁺ ECs of both Tie2-GFPmir142T and Tie2-GFP mice. (B) Flow cytometry analysis of blood cells of representative wild-type (n = 1), Tie2-GFP (n = 2), and Tie2-GFPmir142T (n = 3) mice. Cd11b⁺ myeloid cells are gated (dot plot on the left) and GFP expression analyzed in this hematopoietic subset (dot plots on the right). Note that GFP is expressed in the Cd11b⁺ SSC^low monocytes of Tie2-GFP, but not Tie2-GFPmir142T mice. Ly indicates lymphocytes; Gr, granulocytes; Mo, monocytes. (C-E) Flow cytometry analysis of the blood of Tie2-GFP transgenic mice. (C) Virtually all the Tie2-GFP⁺ TEMs (green gate in the dot plot on the left; n = 16) are Cd11b⁺Cd115⁺7AAD⁻ monocytes (dot plot on the right). (D) Only a fraction (∼ 30%; n = 13) of the Cd11b⁺Cd115⁺7AAD⁻ monocytes (dot plot on the left) are Tie2-GFP⁺ (dot plot in the middle). Monocytes from wild-type mice (n = 5) were used to set the gate for GFP-positive events (dot plot on the right). (E) Dot plots show the expression of surface markers that can distinguish resident from inflammatory monocytes (n = 7-10). The Cd115⁺GFP⁺7AAD⁻ cells were gated and the expression of Cd43, Gr1, and L-selectin (Cd62l) analyzed. The majority of Tie2-GFP⁺Cd115⁺7AAD⁻ TEMs are Cd43⁺Gr1⁻Cd62l⁻ resident monocytes. Representative experiments are shown.