To track the generation and degradation of PIPs, and to determine PIP-metabolizing enzymes, one can incubate phagosomes with PIP sensors and ATP at a physiological temperature, followed by the use of specific inhibitors.
The engulfment of large particles by professional phagocytic cells, like macrophages, occurs within a specific endocytic compartment, the phagosome. This phagosome subsequently fuses with a lysosome, transforming into a phagolysosome, ultimately leading to the degradation of the engulfed materials. Phagosome maturation is regulated by the progressive merging of the phagosome, first with early sorting endosomes, then with late endosomes, and finally with lysosomes. Vesicle fission from the maturing phagosome, coupled with the dynamic on-and-off cycles of cytosolic proteins, causes subsequent alterations. A detailed protocol for reconstituting fusion events between phagosomes and different endocytic compartments is presented within a cell-free system. This reconstitution method serves to delineate the identities of, and the intricate relationships between, pivotal figures in the fusion events.
To preserve the body's equilibrium and protect it from infection, the process of immune and non-immune cells ingesting self and non-self particles is critical. Engulfed particles are found inside phagosomes, vesicles which undergo dynamic fusion and fission. This results in the formation of phagolysosomes, which digest the contained cargo. Homeostasis is maintained by this highly conserved process, and its disruption is implicated in a variety of inflammatory ailments. Given phagosomes' critical function within innate immunity, a deeper understanding of how diverse cellular stimuli and internal changes can impact their architectural design is paramount. This chapter describes a robust procedure for the isolation of polystyrene bead-induced phagosomes, employing the technique of sucrose density gradient centrifugation. This process leads to the production of a sample of exceptional purity, applicable in subsequent processes, including Western blotting.
The process of phagocytosis culminates in a newly defined, terminal stage known as phagosome resolution. During this period, phagolysosomes undergo a process of fragmentation, resulting in the formation of smaller vesicles that we have named phagosome-derived vesicles (PDVs). Macrophages hold an increasing amount of PDVs, and phagosomes shrink in size until these intracellular organelles become imperceptible. The maturation markers of PDVs align with those of phagolysosomes, yet their diverse sizes and highly dynamic nature present a significant impediment to their tracking. Accordingly, to study PDV populations inside cells, we developed methods for separating PDVs from the phagosomes from whence they originated, and then to further characterize their attributes. Two microscopy-based methods, described in this chapter, allow for the quantification of phagosome resolution aspects, such as volumetric analysis of phagosome shrinkage and PDV accumulation, and the analysis of co-occurrence patterns between diverse membrane markers and PDVs.
To facilitate its pathogenic actions, Salmonella enterica serovar Typhimurium (S.) needs to establish an intracellular locale within mammalian cells. The significance of Salmonella Typhimurium as a pathogen should not be underestimated. The gentamicin protection assay methodology will be presented for the study of Salmonella Typhimurium internalization in human epithelial cells. The assay strategically uses gentamicin's limited penetration into mammalian cells to protect internalized bacteria from its antibacterial effects. A second assay, the chloroquine (CHQ) resistance assay, is employed to gauge the portion of internalized bacteria whose Salmonella-containing vacuole has been lysed or compromised, causing them to be located within the cytosol. The presentation will also include its application to quantify cytosolic S. Typhimurium present within epithelial cells. Using these protocols, a quantitative assessment of S. Typhimurium's bacterial internalization and vacuole lysis is rapid, sensitive, and inexpensive.
Phagosome maturation, alongside phagocytosis, are central to the progression of both the innate and adaptive immune response. Invertebrate immunity A rapid and continuous, dynamic process is phagosome maturation. Quantitative and temporal analyses of phagosome maturation, focusing on beads and M. tuberculosis as phagocytic targets, are described in this chapter using fluorescence-based live cell imaging methods. We describe, as well, simple procedures for the monitoring of phagosome maturation, relying on the acidotropic dye LysoTracker, and the examination of host protein recruitment to phagosomes, which are tagged with EGFP.
A key role in macrophage-mediated inflammation and homeostasis is played by the phagolysosome, a specialized organelle with both antimicrobial and degradative properties. The presentation of phagocytosed proteins to the adaptive immune system depends on their prior processing into immunostimulatory antigens. The significance of other processed PAMPs and DAMPs stimulating an immune response, if isolated inside the phagolysosome, has only come into sharp focus recently. The mature phagolysosome, within macrophages, releases partially digested immunostimulatory PAMPs and DAMPs, a process known as eructophagy, to activate nearby leukocytes, through an extracellular pathway. The chapter systematically outlines methods for observing and quantifying eructophagy, involving the simultaneous measurement of multiple parameters associated with each phagosome. These methods, incorporating real-time automated fluorescent microscopy, utilize specifically designed experimental particles capable of bonding to multiple reporter/reference fluors. High-content image analysis software provides the capacity to evaluate each phagosomal parameter either quantitatively or semi-quantitatively in the post-analysis stage.
The ability of dual-wavelength, dual-fluorophore ratiometric imaging to assess pH inside cellular compartments has proven to be exceptionally helpful. Dynamic live-cell imaging is facilitated, factoring in changes in focal plane, differences in fluorescent probe loading, and photobleaching from repeated image capture. Ratiometric microscopic imaging provides the unique capability of resolving individual cells and organelles, an improvement over whole-population methods. find more A detailed discourse on ratiometric imaging and its application to the measurement of phagosomal pH, including probe selection, instrumental needs, and calibration methods, is presented in this chapter.
In the context of organelles, the phagosome is redox-active. Reductive and oxidative systems are essential for phagosomal activity, both directly and indirectly. Redox conditions within the maturing phagosome, their regulation, and their effects on other phagosomal functions can now be investigated with the introduction of newer live-cell techniques to study these redox events. This chapter details real-time, fluorescence-based assays for measuring disulfide reduction and reactive oxygen species production in live phagocytes, including macrophages and dendritic cells, focusing on phagosome-specific mechanisms.
Macrophages and neutrophils, among other cells, internalize a diverse array of particulate matter, including bacteria and apoptotic bodies, via the process of phagocytosis. Phagosomes encapsulate these particles, subsequently merging with early and late endosomes, and finally with lysosomes, thereby achieving phagolysosome maturation through the process of phagosome maturation. Particle degradation ultimately results in phagosome fragmentation, a critical step in the reformation of lysosomes through the mechanism of phagosome resolution. The distinct phases of phagosome maturation and resolution are marked by the recruitment and release of proteins that contribute to the development and eventual clearance of the phagosome. The evaluation of these changes at the single-phagosome level is achievable via immunofluorescence methods. Phagosome maturation is often tracked using indirect immunofluorescence techniques, these methods relying on primary antibodies targeting specific molecular markers. The identification of phagolysosome formation from phagosomes is frequently accomplished by staining cells with antibodies targeting Lysosomal-Associated Membrane Protein I (LAMP1) and measuring the fluorescence intensity of LAMP1 around each phagosome through microscopy or flow cytometry. genetic stability However, the application of this method extends to any molecular marker possessing immunofluorescence-compatible antibodies.
Hox-driven conditionally immortalized immune cells have seen a substantial rise in biomedical research applications over the past fifteen years. The capacity of myeloid progenitor cells, conditionally immortalized by HoxB8, to differentiate into operational macrophages is preserved. This conditional immortalization strategy yields numerous advantages, including limitless propagation, genetic variability, on-demand access to primary-like immune cells (macrophages, dendritic cells, and granulocytes), derivation from a diverse range of mouse strains, and simple cryopreservation and reconstitution procedures. The derivation and application of HoxB8-immortalized myeloid progenitor cells are explained in this chapter.
Filamentous targets become internalized by phagocytic cups, which persist for several minutes before they constrict, culminating in phagosome formation. This attribute's benefit is to facilitate studies of significant phagocytosis events with a more precise spatial and temporal resolution than methods using spherical particles allow. The rapid formation of the phagosome from the phagocytic cup occurs within just a few seconds of the particle being attached. The chapter introduces methods for cultivating filamentous bacteria and elucidates their use as subjects for research into the phagocytic process.
The motile and morphologically adaptable nature of macrophages hinges on significant cytoskeletal restructuring to execute their pivotal roles in innate and adaptive immunity. Macrophages are exceptionally capable of producing diverse actin-based structures and actions, such as podosome development and phagocytosis, to effectively ingest particles and absorb substantial extracellular fluid volumes through micropinocytosis.