A Real Time Chemotaxis Assay Unveils Unique Migratory Profiles amongst Different Primary Murine Macrophages

Chemotaxis assays are an invaluable tool for studying the biological activity of inflammatory mediators such as CC chemokines, which have been implicated in a wide range of chronic inflammatory diseases. Conventional chemotaxis systems such as the modified Boyden chamber are limited in terms of the data captured given that the assays are analysed at a single time-point. We report the optimisation and validation of a label-free, real-time cell migration assay based on electrical cell impedance to measure chemotaxis of different primary murine macrophage populations in response to a range of CC chemokines and other chemoattractant signalling molecules. We clearly demonstrate key differences in the migratory behavior of different murine macrophage populations and show that this dynamic system measures true macrophage chemotaxis rather than chemokinesis or fugetaxis. We highlight an absolute requirement for Gαi signaling and actin cytoskeletal rearrangement as demonstrated by Pertussis toxin and cytochalasin D inhibition. We also studied the chemotaxis of CD14+ human monocytes and demonstrate distinct chemotactic profiles amongst different monocyte donors to CCL2. This real-time chemotaxis assay will allow a detailed analysis of factors that regulate macrophage responses to chemoattractant cytokines and inflammatory mediators.

趋化试验（chemotaxis assay）是研究炎症介质生物学活性的宝贵工具，这类介质包括CC趋化因子（CC chemokines），后者与多种慢性炎症性疾病密切相关。传统趋化系统（如改良博伊登小室）存在数据捕获局限，原因在于此类试验仅能在单时间点进行分析。本研究报道了一种基于细胞电阻抗的无标记实时细胞迁移试验的优化与验证方案，该试验可用于检测不同原代小鼠巨噬细胞群体对多种CC趋化因子及其他趋化信号分子的趋化活性。本研究明确证实了不同小鼠巨噬细胞群体的迁移行为存在显著差异，并证明该动态系统可检测真实的巨噬细胞趋化性，而非趋激性（chemokinesis）或趋退性（fugetaxis）。本研究证实，Gαi信号通路与肌动蛋白细胞骨架重排是巨噬细胞趋化的必需条件，这一结论通过百日咳毒素与细胞松弛素D的抑制实验得到了验证。本研究同时探究了CD14阳性人单核细胞（CD14+ human monocytes）的趋化活性，并证实不同供体的单核细胞对CC趋化因子配体2（CCL2）具有截然不同的趋化特征。该实时趋化试验可用于深入分析调控巨噬细胞对趋化细胞因子及炎症介质应答的各类因素。