Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Abstract

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. Video LinkThe video component of this article can be found at

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator

“…In conclusion, there are other methods available to measure phagosomal pH using different fluorescent dyes as Nunes et al . have demonstrated 13 as well as other groups 27 28 . Other researchers have also used S-1 to measure cytosolic pH 29 or phagosomal pH 14 .…”

“…A further significant problem with FITC in the context of neutrophils is that it is thought to be photobleached by MPO 12 . The MPO inhibitor, sodium azide, can be used to limit photobleaching 13 , but it has been shown that sodium azide directly lowers the phagosomal pH in an MPO-independent manner and is thus inappropriate for use in such assays 5 .…”

UNC93B1 interacts with the calcium sensor STIM1 for efficient antigen cross-presentation in dendritic cells

“…To determine whether the decrease in OVA degradation in 3d/3d phagosomes reflected a change in endosomal/phagosomal acidification, we measured endosomal pH at 60 min after DCs incubation with 40 kDa dextrans coupled to pH-sensitive (FITC) or to pH-insensitive dyes (Alexa-647) 16 . Phagosomal pH was quantified via ratiometric imaging by exposing cells to FITC-coupled OVA-coated zymosan, as previously described 28 , 29 . Both endosomes and phagosomes from 3d/3d BM-DCs had increased pH compared to WT cells (Fig.…”

Phase transitions during cyclic inflation and non-Gaussianity

“…Of course, if the universe is locked in the negative potential energy phase, it will keep on inflating. However, as shown in [2], depending upon the nature of the potential, one can exit primordial inflation through a classical transition from negative to positive potential regions, see also [3][4][5][6] for the study and implementation of similar mechanisms. To our knowledge, CI model was the first to realize a phenomenologically successful cosmology with negative potential energy 1 , and may have interesting implications * Electronic address: tbiswas@loyno.edu † Electronic address: tomik@astro.uio.no ‡ Electronic address: a.mazumdar@lancaster.ac.uk 1 For more recent efforts in this direction see [7], though ghost and tachyonic instabilities may develop in this proposal [8].…”

“…3 In general the infrared divergence of quantum fluctuations that could otherwise dominate over the thermal fluctuations may also be resolved in nonsingular cosmologies [26]. 4 For some of the other attempts to resolve the Big Bang/Big Crunch singularity through bounces when a high energy scale is reached, see [27][28][29][30][31][32][33].…”