Foote Juliet R, Levine Adam P, Behe Philippe, Duchen Michael R, Segal Anthony W
Centre for Molecular Medicine, Division of Medicine, University College London.
Cell and Developmental Biology, University College London.
J Vis Exp. 2017 Apr 5(122):55107. doi: 10.3791/55107.
Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement.
中性粒细胞对宿主固有防御至关重要，因此构成了医学研究的一个重要领域。吞噬体是细胞内进行吞噬颗粒杀伤和消化的区室，是中性粒细胞杀灭病原体的主要场所，这一过程需要严格调控。吞噬体pH是受到精细控制的一个方面，进而调节抗菌蛋白酶的活性。许多荧光pH敏感染料已被用于观察吞噬体环境。与其他pH敏感染料相比，S-1具有多个优点，包括其双发射光谱、抗光漂白性以及高pKa值。使用这种方法，我们已经证明，与其他吞噬细胞相比，中性粒细胞吞噬体的碱性异常。通过使用染料的不同生化偶联物，可以将吞噬体与细胞质区分开来，从而能够评估吞噬体大小和形状的变化。这有助于进一步监测离子运动。
