A Simple and Effective Flow Cytometry-Based Method for Identification and Quantification of Tissue Infiltrated Leukocyte Subpopulations in a Mouse Model of Peripheral Arterial Disease

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+ 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.

Rag1 Deficiency Impairs Arteriogenesis in Mice

“…Secondly, M2-like polarized regenerative macrophages, which are identifiable through the presence of MRC1 [ 21 , 22 ]. Analyzing spatial and temporal macrophage dynamics revealed that the magnitude of arteriogenesis is directly proportional to the optimal balance of M1-like and M2-like macrophages [ 23 ].…”

“…Arteriogenesis was induced by femoral artery ligation (FAL) according to the previously published protocol [ 23 , 32 ]. Briefly, after anesthetizing mice with a standard combination of fentanyl (0.05 mg/kg; CuraMED, Karlsruhe, Germany), midazolam (5.0 mg/kg; Ratiopharm, Ulm, Germany), and medetomidine (0.5 mg/kg; Pfister Pharma, Berlin, Germany), the skin of the upper thigh on both limbs was disinfected, and hair from the surgical site was shaved.…”

p38 MAPK priming boosts VSMC proliferation and arteriogenesis by promoting PGC1α-dependent mitochondrial dynamics

“…Mice were sacrificed 7 days after HLI surgery by means of CO2 and perfused with 10 ml of cold PBS. Surface adductors were dissected as previously described 86 and collected in cold DMEM. Next, adductor muscles were diced and digested in 0.1% collagenase IV (C5138, Sigma) for 45 min at 37 °C.…”

From Increased Fluid Shear Stress to Natural Bypass Growth

“…Co-workers of the group of Elisabeth Deindl describe the functional role of extracellular RNA in that process and highlight the role of this nucleic acid during ongoing arteriogenesis [3]. Moreover, the same group presents a simple flow cytometry-based method to identify and quantify tissue infiltrated leukocyte subpopulations [4]. Macrophages, which accumulate in the perivascular tissue of growing collateral arteries, are well described for their pro-arteriogenic feature.…”