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Protocol for analysis of mouse neutrophil NETosis by flow cytometry

ABSTRACT: Summary Studies involving neutrophils are steadily increasing, thus creating a need for more optimized and thorough protocols for studying neutrophil function. Here, we present our protocol for extracting mouse bone marrow neutrophils, estimating the purity of isolated neutrophils, and assessing their ability to induce NETosis upon an external cue. We test two isolation protocols that can be used to attain neutrophils to assess NETosis induction. This approach allows for the parallel assessment of NETosis induction in cohorts larger than 10 samples. For complete details on the use and execution of this protocol, please refer to Lu et al., 2021. Graphical abstract Highlights • Primary neutrophil isolation and functional analysis can be completed within a day• Neutrophil NETosis variations can be examined across sex, age, genotype, and treatment• NETosis induction can be assessed in many biological samples (>10) in parallel Studies involving neutrophils are steadily increasing, thus creating a need for more optimized and thorough protocols for studying neutrophil function. Here, we present our protocol for extracting mouse bone marrow neutrophils, estimating the purity of isolated neutrophils, and assessing their ability to induce NETosis upon an external cue. We test two isolation protocols that can be used to attain neutrophils to assess NETosis induction. This approach allows for the parallel assessment of NETosis induction in cohorts larger than 10 samples.

SUBMITTER: McGill C

PROVIDER: S-EPMC8599168 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Flow cytometry is a powerful method, which is widely used for high-throughput quantitative and qualitative analysis of cells. However, its straightforward applicability for extracellular vesicles (EVs) and mainly exosomes is hampered by several challenges, reflecting mostly the small size of these vesicles (exosomes: ~80-200 nm, microvesicles: ~200-1,000 nm), their polydispersity, and low refractive index. The current best and most widely used protocol for beads-free flow cytometry of exosomes uses ultracentrifugation (UC) coupled with floatation in sucrose gradient for their isolation, labeling with lipophilic dye PKH67 and antibodies, and an optimized version of commercial high-end cytometer for analysis. However, this approach requires an experienced flow cytometer operator capable of manual hardware adjustments and calibration of the cytometer. Here, we provide a novel and fast approach for quantification and characterization of both exosomes and microvesicles isolated from cell culture media as well as from more complex human samples (ascites of ovarian cancer patients) suitable for multiuser labs by using a flow cytometer especially designed for small particles, which can be used without adjustments prior to data acquisition. EVs can be fluorescently labeled with protein-(Carboxyfluoresceinsuccinimidyl ester, CFSE) and/or lipid- (FM) specific dyes, without the necessity of removing the unbound fluorescent dye by UC, which further facilitates and speeds up the characterization of microvesicles and exosomes using flow cytometry. In addition, double labeling with protein- and lipid-specific dyes enables separation of EVs from common contaminants of EV preparations, such as protein aggregates or micelles formed by unbound lipophilic styryl dyes, thus not leading to overestimation of EV numbers. Moreover, our protocol is compatible with antibody labeling using fluorescently conjugated primary antibodies. The presented methodology opens the possibility for routine quantification and characterization of EVs from various sources. Finally, it has the potential to bring a desired level of control into routine experiments and non-specialized labs, thanks to its simple bead-based standardization.

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Protocol for analysis of mouse neutrophil NETosis by flow cytometry

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