Walter-Brendel-Centre of Experimental Medicine, University Hospital, LMU Munich, 81377 Munich, Germany.
Biomedical Center, Institute of Cardiovascular Physiology and Pathophysiology, LMU Munich, 82152 Planegg-Martinsried, Germany.
Int J Mol Sci. 2020 May 19;21(10):3593. doi: 10.3390/ijms21103593.
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.
动脉生成，即从预先存在的小动脉侧支生长出自然旁路，是一种补偿动脉损失的内源性机制。从机制上讲，这个过程依赖于白细胞亚群的局部和暂时的血管周围浸润，这些白细胞亚群通过提供生长因子和细胞因子来介导动脉生成。目前，在小鼠模型中识别和定量这些白细胞亚群的最新方法是免疫组织化学。然而，这是一个耗时的过程。在这里，我们旨在开发一种优化的方案，通过血流细胞术在含有生长性侧支动脉的收肌中识别和定量白细胞亚群。为此，在动脉生成诱导后第 1 天和第 3 天分离小鼠后肢的收肌，通过酶消化，然后通过流式细胞术识别和定量白细胞亚群，如图中所示，示例为中性粒细胞和巨噬细胞（分别定义为 CD45/CD11b/Ly6G 和 CD45/CD11b/F4/80 细胞）。总之，我们表明，流式细胞术是一种在肌肉组织中识别和定量白细胞亚群的合适方法，并提供了详细的方案。与组织学相比，流式细胞术是一种节省时间的工具，它可以与组织学结合使用，以更精确地定位组织样本中的白细胞。
