دانلود رایگان مقاله پلت فرم تصویربرداری جدید زمان واقعی برای تعیین کمیت فاگوسیتوز ماکروفاژها

abstract

Phagocytosis of pathogens, apoptotic cells and debris is a key feature of macrophage function in host defense and tissue homeostasis. Quantification of macrophage phagocytosis in vitro has traditionally been technically challenging. Here we report the optimization and validation of the IncuCyte ZOOM
real time imaging platform for macrophage phagocytosis based on pHrodo
pathogen bioparticles, which only fluoresce when localized in the acidic environment of the phagolysosome. Image analysis and fluorescence quantification were performed with the automated IncuCyteTM Basic Software. Titration of the bioparticle number showed that the system is more sensitive than a spectrofluorometer, as it can detect phagocytosis when using 20
less E. coli bioparticles. We exemplified the power of this real time imaging platform by studying phagocytosis of murine alveolar, bone marrow and peritoneal macrophages. We further demonstrate the ability of this platform to study modulation of the phagocytic process, as pharmacological inhibitors of phagocytosis suppressed bioparticle uptake in a concentration-dependent manner, whereas opsonins augmented phagocytosis. We also investigated the effects of macrophage polarization on E. coli phagocytosis. Bone marrow-derived macrophage (BMDM) priming with M2 stimuli, such as IL-4 and IL-10 resulted in higher engulfment of bioparticles in comparison with M1 polarization. Moreover, we demonstrated that tolerization of BMDMs with lipopolysaccharide (LPS) results in impaired E. coli bioparticle phagocytosis. This novel real time assay will enable researchers to quantify macrophage phagocytosis with a higher degree of accuracy and sensitivity and will allow investigation of limited populations of primary phagocytes in vitro.

4. Discussion

In this study we describe a novel imaging platform to quantify macrophage phagocytosis in vitro. This technology combines pHdependent dye particle labeling, the acquisition of images in real time and operator-independent image analysis. We have exempli- fied the quantitative aspect of this technology platform using drugs that inhibit cytoskeletal changes and lysosome acidification. Furthermore, we show the ability to quantify enhancement of phagocytosis in primary macrophages using repeated measures every 10 min. We hereby show that this technology can be successfully applied to overcome common technical limitations when carrying out phagocytosis assays in vitro. Foreign object phagocytosis has traditionally been studied as the uptake of microspheres, fluorescent pathogens or sheep erythrocytes. The advantage of using the commercially available pHrodo pathogen bioparticles is that they only fluoresce when they are localized in an acidic micro-environment, like that of the phagolysosome. Our data clearly demonstrate that pHrodolabeled bioparticles do not fluoresce when bound to the macro phage membrane or when free in the culture supernatant, in contrast to latex beads or FITC-labeled pathogens. Microscopy [26,27] and flow cytometry techniques [28] have been used to visualize and quantify phagocytosis of E. coli bioparticles by non-adherent phagocytic cells or macrophage cell lines. However, primary macrophages can be stressed when detached from plastic to be run on a flow or imaging flow cytometer and quantification of phagocytosis from microscopy image analysis is of low throughput and potentially open to operator bias. The platform we present here uses microscopy to capture and quantify primary murine macrophage phagocytosis and has no negative impact on cell viability.