Maeß Marten B, Wittig Berith, Lorkowski Stefan
Institute of Nutrition, Friedrich Schiller University Jena.
Institute of Nutrition, Friedrich Schiller University Jena;
J Vis Exp. 2014 Sep 2(91):e51960. doi: 10.3791/51960.
Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. Transfection with siRNA and plasmid DNA is an efficient tool for studying their function, but transfection of macrophages is not a trivial matter. Although many different approaches for transfection of eukaryotic cells are available, only few allow reliable and efficient transfection of macrophages, but reduced cell vitality and severely altered cell behavior like diminished capability for differentiation or polarization are frequently observed. Therefore a transfection protocol is required that is capable of transferring siRNA and plasmid DNA into macrophages without causing serious side-effects thus allowing the investigation of the effect of the siRNA or plasmid in the context of normal cell behavior. The protocol presented here provides a method for reliably and efficiently transfecting human THP-1 macrophages and monocytes with high cell vitality, high transfection efficiency, and minimal effects on cell behavior. This approach is based on Nucleofection and the protocol has been optimized to maintain maximum capability for cell activation after transfection. The protocol is adequate for adherent cells after detachment as well as cells in suspension, and can be used for small to medium sample numbers. Thus, the method presented is useful for investigating gene regulatory effects during macrophage differentiation and polarization. Apart from presenting results characterizing macrophages transfected according to this protocol in comparison to an alternative chemical method, the impact of cell culture medium selection after transfection on cell behavior is also discussed. The presented data indicate the importance of validating the selection for different experimental settings.
巨噬细胞作为先天性免疫反应的关键参与者，是研究组织稳态或各种病理状态的焦点。用小干扰RNA（siRNA）和质粒DNA进行转染是研究其功能的有效工具，但巨噬细胞的转染并非易事。尽管有许多不同的真核细胞转染方法，但只有少数方法能实现对巨噬细胞可靠且高效的转染，而且经常会观察到细胞活力降低以及细胞行为严重改变，如分化或极化能力减弱。因此，需要一种转染方案，能够将siRNA和质粒DNA导入巨噬细胞而不产生严重的副作用，从而在正常细胞行为的背景下研究siRNA或质粒的作用。本文介绍的方案提供了一种可靠且高效地转染人THP-1巨噬细胞和单核细胞的方法，具有高细胞活力、高转染效率，且对细胞行为影响最小。该方法基于核转染技术，并且该方案已进行优化，以在转染后维持最大的细胞激活能力。该方案适用于贴壁细胞脱落后以及悬浮细胞，可用于中小规模样本。因此，所介绍的方法对于研究巨噬细胞分化和极化过程中的基因调控作用很有用。除了展示根据该方案转染的巨噬细胞与另一种化学方法相比的特征结果外，还讨论了转染后细胞培养基选择对细胞行为的影响。所呈现的数据表明了针对不同实验设置验证选择的重要性。
