Resolution of inflammation is an important physiological process following infection or injury. When inflammation fails to resolve, it can cause chronic inflammation, which exacerbates a myriad of diseases. Current anti-inflammatory treatment options are often inadequate to resolve inflammation, and as such, a key goal for drug discovery is to find natural products and novel compounds that can target immune resolution processes. In order to efficiently discovery new therapies, immune cell lines are often used, in conjunction with flow cytometry, to quickly and inexpensively screen potential drugs for immunomodulatory effects. However, seemingly minor or trivial differences in methodology can lead to inconsistent results across experiments and across laboratories. It was the goal of this project to examine the effects of those differences on the RAW 264.7 macrophage cell line, particularly as it relates to macrophage polarization experimentation. We found that the type of detachment method when preparing cells for flow cytometry can alter several key macrophage parameters, including markers for macrophage polarization, depending on the gating strategy used in identifying sub-populations of cells for analysis. Investigators need to incorporate best-practices in gating strategy in order to target viable cells that are not in aggregate to ensure consistent and reliable results for immunomodulatory drug discovery.

炎症消退是感染或受伤后的一个重要生理过程。当炎症无法消退时，会导致慢性炎症，从而加剧多种疾病。当前的抗炎治疗方案通常不足以解决炎症，因此，药物发现的一个关键目标是找到可以针对免疫解决过程的天然产物和新型化合物。为了有效地发现新疗法，免疫细胞系通常与流式细胞术结合使用，以快速且廉价地筛选具有免疫调节作用的潜在药物。然而，方法学上看似微小或微不足道的差异可能会导致不同实验和实验室的结果不一致。该项目的目标是检查这些差异对 RAW 264.7 巨噬细胞系的影响，特别是因为它与巨噬细胞极化实验有关。我们发现，在为流式细胞术准备细胞时，分离方法的类型可以改变几个关键的巨噬细胞参数，包括巨噬细胞极化的标记，这取决于用于识别细胞亚群进行分析的门控策略。研究人员需要将最佳实践纳入门控策略，以针对非聚集的活细胞，以确保免疫调节药物发现的结果一致和可靠。我们发现，在为流式细胞术准备细胞时，分离方法的类型可以改变几个关键的巨噬细胞参数，包括巨噬细胞极化的标记，这取决于用于识别细胞亚群进行分析的门控策略。研究人员需要将最佳实践纳入门控策略，以针对非聚集的活细胞，以确保免疫调节药物发现的结果一致和可靠。我们发现，在为流式细胞术准备细胞时，分离方法的类型可以改变几个关键的巨噬细胞参数，包括巨噬细胞极化的标记，这取决于用于识别细胞亚群进行分析的门控策略。研究人员需要将最佳实践纳入门控策略，以针对非聚集的活细胞，以确保免疫调节药物发现的结果一致和可靠。