Systematic approach in macrophage polarization experiments: Maintaining integrity and reproducibility using flow cytometry and sample preparation.

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.