The ability of phagosomes to stop pathogen growth is closely related to their ability to acidify the pH of their lumen. Our engineers have developed strategies to assess the degree of acidification of phagosomes to analyze protein function during phagosome maturation. Here, CD BioSciences offers specialized phagosome pH measurement services to support the assessment of phagosome acidification.
Background of Measurement of Phagosomal pH
Phagocytosis is the process by which innate immune cells engulf particles. They bind to the target and coat it with a membrane, which is then pinch-off to form a phagosome within the cytoplasm. The intraluminal pH of the phagosome has profound effects on phagosome function, including trafficking in the inner membrane, ionic composition of the phagosome lumen, and proteolytic enzyme activity. Many pathogens, such as Mycobacterium tuberculosis and Salmonella typhi, target the host cell's pH-regulating machinery in an effort to survive within or escape from the phagosome. Importantly, some cytokines may generate signaling events that induce maturation and changes in phagocytic behavior, which in turn affect phagosome pH. Therefore, various cell biologists, immunologists, microbiologists, and drug developers hope to explore the mechanisms underlying the influence of microorganisms on immune responses by measuring phagosomal pH.
Fig. 1. Regulation of phagosomal pH in phagocytes. (Westman J, et al., 2021)
Phagosomal pH Measurement Services
Many intracellular pathogens disrupt phagosome killing by limiting phagosome acidification, demonstrating the importance of pH in phagosome biology. CD BioSciences focuses on the measurement of phagosome pH to assess the degree of phagosome acidification. We developed efficient methods to measure the average pH of phagosome populations. We chose fluorescein isothiocyanate (FITC) as a pH-sensitive dye, and assessed phagosome, efferosome or endosome acidification in any cell type using any target that can be conjugated to FITC.
Our method can be easily modified to measure pH across different phagocytic cell types and is suitable for utilizing other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a general protocol for imaging phagosome function.
- Preparation of phagocytic targets.
We tracked it using wide-field fluorescence microscopy, where cells were imaged as they phagocytosed, and pH changes in phagosomes were measured in real time as they matured. - Perform a calibration experiment to collect the actual pH value.
- Analysis of time-lapse movies and phagosome snapshots.
- Compare the ratio value with the known pH value of the solution.
We constructed a calibration curve by nonlinear regression and used the resulting equation to calculate pH from ratio values.
Our Advantages
- FITC is inexpensive and widely available, with a pKa of 6.5 and a dynamic range of its free form including pH 3-9.
- Internally and externally bound but uninternalized particles can be easily distinguished without the addition of additional chemicals.
- Tracking cells in real time allows researchers to observe multiple aspects of the phagocytosis process simultaneously, easily discerning effects on migration, sensing and binding of particles.
We look forward to collaborating with you. You are always welcome to engage in discussions with us at any point of the project. If you are interested in our services, please feel free to contact us for more information.
Reference
- Westman J, Grinstein S. (2021) Determinants of phagosomal pH during host-pathogen interactions[J]. Frontiers in cell and developmental biology. 8: 624958.