*Communication*

### **A Simple and E**ff**ective Flow Cytometry-Based Method for Identification and Quantification of Tissue Infiltrated Leukocyte Subpopulations in a Mouse Model of Peripheral Arterial Disease**

**Konda Kumaraswami 1,2, Natallia Salei 1,2, Sebastian Beck 1,2, Stephan Rambichler 1,2, Anna-Kristina Kluever 1,2, Manuel Lasch 1,2,3, Lisa Richter 4, Barbara U. Schraml 1,2 and Elisabeth Deindl 1,2,\***


Received: 6 May 2020; Accepted: 16 May 2020; Published: 19 May 2020

**Abstract:** Arteriogenesis, the growth of a natural bypass from pre-existing arteriolar collaterals, is an endogenous mechanism to compensate for the loss of an artery. Mechanistically, this process relies on a locally and temporally restricted perivascular infiltration of leukocyte subpopulations, which mediate arteriogenesis by supplying growth factors and cytokines. Currently, the state-of-the-art method to identify and quantify these leukocyte subpopulations in mouse models is immunohistology. However, this is a time consuming procedure. Here, we aimed to develop an optimized protocol to identify and quantify leukocyte subpopulations by means of flow cytometry in adductor muscles containing growing collateral arteries. For that purpose, adductor muscles of murine hindlimbs were isolated at day one and three after induction of arteriogenesis, enzymatically digested, and infiltrated leukocyte subpopulations were identified and quantified by flow cytometry, as exemplary shown for neutrophils and macrophages (defined as CD45+/CD11b+/Ly6G+ and CD45+/CD11b+/F4/80<sup>+</sup> cells, respectively). In summary, we show that flow cytometry is a suitable method to identify and quantify leukocyte subpopulations in muscle tissue, and provide a detailed protocol. Flow cytometry constitutes a timesaving tool compared to histology, which might be used in addition for precise localization of leukocytes in tissue samples.

**Keywords:** peripheral arterial disease; arteriogenesis; shear stress; flow cytometry; leukocytes; immunohistology; inflammation; collateral artery growth
