Quantitative Live-Cell Fluorescence Microscopy During Phagocytosis

Lu, Stella M.; Grinstein, Sergio; Fairn, Gregory D.

doi:10.1007/978-1-4939-6581-6_6

Cited by 10 publications

(7 citation statements)

References 26 publications

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“…Given the dynamic nature of these events, the preferred method for the detection of phagosomal tubulation is live-cell imaging [24,98,99]. As a less efficient but in some cases more accessible alternative, phagotubules may also be visualized on fixed cells by IF or immunoelectron microscopy [27].…”

Section: Imaging and Fluorescence-based Methodsmentioning

confidence: 99%

A guide to measuring phagosomal dynamics

Levin‐Konigsberg

Mantegazza

2020

The FEBS Journal

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Phagocytosis is an essential mechanism for immunity and homeostasis, performed by a subset of cells known as phagocytes. Upon target engulfment, de novo formation of specialized compartments termed phagosomes takes place. Phagosomes then undergo a series of fusion and fission events as they interact with the endolysosomal system and other organelles, in a dynamic process known as phagosome maturation. Because phagocytes play a key role in tissue patrolling and immune surveillance, phagosome maturation is associated with signaling pathways that link phagocytosis to antigen presentation and the development of adaptive immune responses. In addition, and depending on the nature of the cargo, phagosome integrity may be compromised, triggering additional cellular mechanisms including inflammation and autophagy. Upon completion of maturation, phagosomes enter a recently described phase: phagosome resolution, where catabolites from degraded cargo are metabolized, phagosomes are resorbed, and vesicles of phagosomal origin are recycled. Finally, phagocytes return to homeostasis and become ready for a new round of phagocytosis. Altogether, phagosome maturation and resolution encompass a series of dynamic events and organelle crosstalk that can be measured by biochemical, imaging, photoluminescence, cytometric, and immune-based assays that will be described in this guide.

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Section: Imaging and Fluorescence-based Methodsmentioning

confidence: 99%

A guide to measuring phagosomal dynamics

Levin‐Konigsberg

Mantegazza

2020

The FEBS Journal

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“…Imaging FCM allows defining the intracellular localization of fluorescent targets in phagocytes, thus ruling out the need of quenching or blocking steps (Fig. ) .…”

Section: Biological Applicationsmentioning

confidence: 99%

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

et al. 2019

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These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.

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“…Other techniques that have been instrumental for our present understanding of phagocytosis are fluorescence microscopy coupled to particular probes to measure phagosome pH ( 301 ), to describe phospholipid dynamics during phagosome formation ( 302 ), and to quantify antibody-dependent phagocytosis ( 303 ). Together with these, the use of confocal microscopy coupled to fluorescence resonance energy transfer-based assays has been helpful to investigate the mechanisms of L. monocytogenes for phagosome escaping ( 304 ).…”

Section: Future Directionsmentioning

confidence: 99%

Control of Phagocytosis by Microbial Pathogens

2017

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Phagocytosis is a fundamental process of cells to capture and ingest foreign particles. Small unicellular organisms such as free-living amoeba use this process to acquire food. In pluricellular organisms, phagocytosis is a universal phenomenon that all cells are able to perform (including epithelial, endothelial, fibroblasts, etc.), but some specialized cells (such as neutrophils and macrophages) perform this very efficiently and were therefore named professional phagocytes by Rabinovitch. Cells use phagocytosis to capture and clear all particles larger than 0.5 µm, including pathogenic microorganisms and cellular debris. Phagocytosis involves a series of steps from recognition of the target particle, ingestion of it in a phagosome (phagocytic vacuole), maturation of this phagosome into a phagolysosome, to the final destruction of the ingested particle in the robust antimicrobial environment of the phagolysosome. For the most part, phagocytosis is an efficient process that eliminates invading pathogens and helps maintaining homeostasis. However, several pathogens have also evolved different strategies to prevent phagocytosis from proceeding in a normal way. These pathogens have a clear advantage to perpetuate the infection and continue their replication. Here, we present an overview of the phagocytic process with emphasis on the antimicrobial elements professional phagocytes use. We also summarize the current knowledge on the microbial strategies different pathogens use to prevent phagocytosis either at the level of ingestion, phagosome formation, and maturation, and even complete escape from phagosomes.

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Quantitative Live-Cell Fluorescence Microscopy During Phagocytosis

Cited by 10 publications

References 26 publications

A guide to measuring phagosomal dynamics

A guide to measuring phagosomal dynamics

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

Control of Phagocytosis by Microbial Pathogens

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