Department of Bioengineering and Biophysics Program, University of California Berkeley, Berkeley, CA, USA.
UC Berkeley/UC San Francisco Graduate Group in Bioengineering, Berkeley, CA, USA.
Nat Protoc. 2020 Jul;15(7):2230-2246. doi: 10.1038/s41596-020-0330-8. Epub 2020 Jun 19.
Macrophage phagocytosis can be triggered by diverse receptor-ligand interactions to clear pathogens and dead cells from a host. Many ways of assaying phagocytosis exist that utilize a variety of phagocytic targets with different combinations of receptor-ligand interactions, making comparisons difficult. To study how phagocytosis is affected by specific changes to the target surface, we developed an in vitro assay based on reconstituted membrane-coated target particles to which known molecules can be added. The targets are made by coating glass beads with supported lipid bilayers followed by coupling proteins and other ligands of interest. Composition of the lipid bilayer can be varied to bind and orient specific proteins, incorporate signaling and reporter lipids, and control bilayer fluidity. To quantify phagocytosis, the reconstituted target particles are incubated with macrophages in vitro for a defined period of time, imaged with fluorescence microscopy and analyzed with software that measures the amount of target particle fluorescence within each macrophage. A multi-well plate format can be used for multi-parameter studies (e.g., to investigate how phagocytosis is affected by specific receptor-ligand interactions, ligand density, lipid charge, membrane fluidity and other molecular details). As an example, we demonstrate that antibody-dependent phagocytosis is more efficient for targets with fluid membranes than non-fluid membranes. The assay protocol takes approximately 6 h and requires basic molecular biology, mammalian cell culture and fluorescence microscopy skills. This assay can also be used with other phagocytic and non-phagocytic cells to study the individual or collective roles of receptors and ligands in immune effector function.
巨噬细胞吞噬作用可以通过多种受体-配体相互作用触发，以清除宿主中的病原体和死亡细胞。有许多种检测吞噬作用的方法，它们使用不同组合的受体-配体相互作用的各种吞噬作用靶标，这使得比较变得困难。为了研究吞噬作用如何受到靶表面特定变化的影响，我们开发了一种基于重建膜包被靶颗粒的体外测定法，可在其中添加已知分子。这些靶标是通过在玻璃珠上涂覆支撑脂质双层，然后结合蛋白质和其他感兴趣的配体来制备的。脂质双层的组成可以改变以结合和定向特定的蛋白质，掺入信号和报告脂质，并控制双层流动性。为了定量吞噬作用，将重建的靶颗粒与巨噬细胞在体外孵育一段时间，用荧光显微镜成像，并使用测量每个巨噬细胞内靶颗粒荧光量的软件进行分析。可以使用多孔板格式进行多参数研究（例如，研究特定受体-配体相互作用、配体密度、脂质电荷、膜流动性和其他分子细节如何影响吞噬作用）。例如，我们证明对于具有流动膜的靶标，抗体依赖性吞噬作用比非流动膜更有效。该测定法大约需要 6 小时，需要基本的分子生物学、哺乳动物细胞培养和荧光显微镜技能。该测定法也可用于其他吞噬细胞和非吞噬细胞，以研究受体和配体在免疫效应功能中的个体或集体作用。
