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.

巨噬细胞的吞噬作用可以通过多种受体-配体相互作用来触发，以清除宿主中的病原体和死细胞。存在许多分析吞噬作用的方法，这些方法利用了具有吞噬靶标的受体-配体相互作用的不同组合，从而使比较变得困难。为了研究吞噬作用如何受到靶标表面特定变化的影响，我们开发了一种基于体外试验，该试验基于重构的膜包被靶标颗粒，可以向其中添加已知分子。通过用支持的脂质双层包被玻璃珠，然后偶联蛋白和其他目标配体，来制备靶标。脂质双层的组成可以变化，以结合和定向特定蛋白质，结合信号传导和报告脂质，并控制双层流动性。为了量化吞噬作用，将重组后的目标颗粒与巨噬细胞在体外孵育一段确定的时间，用荧光显微镜成像，并用测量每个巨噬细胞内目标颗粒荧光量的软件进行分析。多孔板形式可用于多参数研究（例如，研究吞噬作用如何受特定受体-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的影响）。作为一个例子，我们证明了抗体依赖性吞噬作用对于具有流体膜的靶比非流体膜更有效。该检测方案大约需要6个小时，并且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术。