Nunes Paula, Guido Daniele, Demaurex Nicolas
Department of Cellular Physiology and Metabolism, University of Geneva;
Department of Cellular Physiology and Metabolism, University of Geneva.
J Vis Exp. 2015 Dec 7(106):e53402. doi: 10.3791/53402.
Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H(+) is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.
吞噬作用是一个基本过程，通过该过程，天然免疫细胞吞噬细菌、凋亡细胞或其他外来颗粒，以杀死或中和摄入的物质，或将其作为抗原呈递并启动适应性免疫反应。吞噬体的pH值是调节与内膜的裂变或融合以及蛋白水解酶激活的关键参数，这些事件使吞噬泡成熟为降解性细胞器。此外，H(+)的转运是产生高水平活性氧（ROS）所必需的，而ROS对于有效杀伤和向其他宿主组织发出信号至关重要。许多细胞内病原体通过限制吞噬体酸化来破坏吞噬杀伤作用，突出了pH值在吞噬体生物学中的重要性。在这里，我们描述了一种使用异硫氰酸荧光素（FITC）标记的酵母聚糖作为吞噬靶标，并通过活细胞成像来测量中性粒细胞吞噬体pH值的比率测定法。该测定基于FITC的荧光特性，当在490nm激发时，FITC会被酸性pH淬灭，但在440nm激发时则不会，从而能够定量单一染料的pH依赖性比率，而不是绝对荧光。还提供了进行原位染料校准以及将比率转换为实际pH值的详细方案。单染料比率测定法通常被认为优于单波长或双染料伪比率测定方案，因为它们对诸如漂白、焦点变化、激光变化和不均匀标记等干扰不太敏感，这些干扰会扭曲测量信号。该方法可以轻松修改以测量其他吞噬细胞类型中的pH值，并且酵母聚糖可以被任何其他含胺颗粒替代，从惰性珠子到活微生物。最后，该方法可以进行调整以利用对不同pH范围或其他吞噬体活性敏感的其他荧光探针，使其成为吞噬体功能成像的通用方案。
