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Optimized protocol for the preparation of single cells from cutaneous wounds for flow cytometric cell sorting and analysis of macrophages

机译：优化方案用于制备来自皮肤伤口的单细胞用于流式细胞术细胞分选和巨噬细胞分析

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摘要

The incidence of chronic, non-healing skin wounds is accelerating, largely due to the epidemic of obesity-related Type 2 diabetes. Abnormal inflammation in wounds contributes to delayed healing. During wound repair, blood monocytes are recruited into the wound bed where they differentiate into macrophages that secrete cytokines and regulate subsequent repair events. Because the study of wound macrophages via immunohistochemistry is often unsatisfactory due to nonspecific antibody staining, the ability to isolate and analyze single cells is important for determining the phenotypes of the wound macrophages. In this article, we have expanded upon a protocol originally described by Wilson et al, 2002 [1], and optimized it for isolation of large numbers of viable macrophages from murine skin wounds that are suitable for flow cytometric cell sorting or analysis. Several parameters were found to be critical for improved macrophage yields, including: (1) The proper amount of starting material (skin tissue); (2) The optimal time for addition of Brefeldin A during enzymatic digestion; (3) Revamped guidelines for centrifugation to maximize cell pellet recovery. This optimized protocol could be further modified to perform cell sorting and flow-based immunophenotyping of any cell type involved in wound healing and inflammation.
机译：慢性，非愈合皮肤伤口的发病率正在加速，主要是由于肥胖相关的2型糖尿病的发病。在伤口发炎异常有助于迁延不愈。在创伤修复，血液中的单核细胞被招入伤口床在那里分化成巨噬细胞分泌细胞因子，调节后续维修活动。因为伤口巨噬细胞的免疫组化通过研究通常非特异性抗体染色由于不令人满意的能力来分离和分析单个细胞是用于确定伤口巨噬细胞的表型很重要的。在本文中，我们已经在最初是由Wilson等人，2002 [1]中描述的协议扩展，并且优化其对于大量来自鼠皮肤伤口适合于流式细胞术细胞分选或分析活的巨噬细胞的分离。几个参数被认为是改进的巨噬细胞的产率，包括关键：（1）起始原料（皮肤组织）的适当量; （2）酶消化过程中添加布雷菲德菌素A的最佳时间; （3）离心以最大化细胞沉淀回收修补的准则。这种优化的协议可以被进一步修改为执行细胞分选和基于流的参与伤口愈合和炎症的任何细胞类型的免疫表型。

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