Immunofluorescence and Confocal Microscopy of Neutrophils

La, Allen

doi:10.1007/978-1-59745-467-4_18

Cited by 23 publications

(25 citation statements)

References 17 publications

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“…Isolated human neutrophils were analyzed by confocal microscopy as previously described [50]. In brief, human neutrophils on uncoated glass coverslips were placed in 24-well plates for 30 min at RT.…”

Section: Confocal Microscopymentioning

confidence: 99%

Neutrophil extracellular trap cell death requires both autophagy and superoxide generation

et al. 2010

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Neutrophil extracellular traps (NETs) are extracellular chromatin structures that can trap and degrade microbes. They arise from neutrophils that have activated a cell death program called NET cell death, or NETosis. Activation of NETosis has been shown to involve NADPH oxidase activity, disintegration of the nuclear envelope and most granule membranes, decondensation of nuclear chromatin and formation of NETs. We report that in phorbol myristate acetate (PMA)-stimulated neutrophils, intracellular chromatin decondensation and NET formation follow autophagy and superoxide production, both of which are required to mediate PMA-induced NETosis and occur independently of each other. Neutrophils from patients with chronic granulomatous disease, which lack NADPH oxidase activity, still exhibit PMA-induced autophagy. Conversely, PMA-induced NADPH oxidase activity is not affected by pharmacological inhibition of autophagy. Interestingly, inhibition of either autophagy or NADPH oxidase prevents intracellular chromatin decondensation, which is essential for NETosis and NET formation, and results in cell death characterized by hallmarks of apoptosis. These results indicate that apoptosis might function as a backup program for NETosis when autophagy or NADPH oxidase activity is prevented.

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Section: Confocal Microscopymentioning

confidence: 99%

Neutrophil extracellular trap cell death requires both autophagy and superoxide generation

et al. 2010

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“…The use of these assays provides key insight into the interactions that result in bacterial killing or survival during infection of host cells. The protocols outlined in this article were used to assess the viability of N. gonorrhoeae that is attached to and inside primary human neutrophils, including in different populations of neutrophil phagosomes 5,13,14 . However, these protocols can be applied to assess viability of gram-positive and gram-negative bacteria in professional phagocytes, nonprofessional phagocytes, and protozoa [15][16][17][18][19][20][21][22][23][24] .…”

Section: Introductionmentioning

confidence: 99%

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Johnson

Criss

2013

JoVE

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Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4',6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells. Video LinkThe video component of this article can be found at

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“…Analysis of granule distribution [12] and the composition of small bacterial phagosomes [7–10] is more readily analyzed by, and may require, the enhanced imaging capacity that is available with confocal microscopy. Advantages of confocal microscopy include the ability to zoom in on single cells and the ability to examine cells as sequential (or merged stacks) of thin optical sections.…”

Section: Methodsmentioning

confidence: 99%

“…Using this approach, specific and azurophilic granules can be detected in the vicinity of COZ phagosomes prior to phagosome-granule fusion (Fig. 2) [12]. …”

Section: Methodsmentioning

confidence: 99%

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Immunofluorescence and Confocal Microscopy of Neutrophils

Allen

2014

Methods in Molecular Biology

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Rapid recruitment of neutrophils to sites of infection and their ability to phagocytose and kill microbes is an important aspect of the innate immune response. Challenges associated with imaging of these cells include their short lifespan and small size and the fact that unstimulated cells are nonadherent. In addition, although cytoplasmic granules are plentiful, the abundance of many other organelles is diminished. Here we reprise methods for analysis of resting and activated cells using immunofluorescence and confocal microscopy, including kinetic analysis of phagosome maturation and degranulation, and detection of intraphagosomal superoxide accumulation. We describe approaches for rapid cell fixation and permeabilization that maximize antigen detection and discuss other variables that also affect data interpretation and image quality (such as cell spreading, degranulation, and phagocytosis). Finally, we show that these methods are also applicable to studies of neutrophil interactions with the extracellular matrix.

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Immunofluorescence and Confocal Microscopy of Neutrophils

Cited by 23 publications

References 17 publications

Neutrophil extracellular trap cell death requires both autophagy and superoxide generation

Neutrophil extracellular trap cell death requires both autophagy and superoxide generation

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Immunofluorescence and Confocal Microscopy of Neutrophils

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