A method for high-purity isolation of neutrophil granulocytes for functional cell migration assays
Date
2019Author
Avci, Edibe
Akkaya-Ulum, Yeliz Z.
Yoyen-Ermis, Digdem
Esendagli, Gunes
Balci-Peynircioglu, Banu
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Background: Neutrophil-mediated killing of pathogens is one of the most
significant functions of the primary defense of the host. Neutrophil
activity and migration play a key role in inflammatory conditions. To
gain insights into the interactions between neutrophils and neutrophil
migration-related disorders, a large number of sophisticated methods
have been developed. The technical limitations of isolating highly
purified neutrophil populations, minimizing both cell death and
activation during the isolation process, and the short lifespan of
neutrophils present challenges for studying specific functions of
neutrophils in vitro. In this study, we aimed to evaluate a separation
medium-based density gradient method to obtain highly purified
neutrophil populations and combined this protocol with a model for
studying neutrophil migration in-vitro.
Materials and methods: Human granulocytes were isolated using
Lympholyte-poly solution. The purity and viability of isolated
neutrophils were assessed by flow cytometry and morphological analysis.
Neutrophil activation was confirmed by immunocytochemistry. Lastly,
filter assay was performed to measure neutrophil chemotaxis.
Results and discussion: All validation experiments revealed that this
method was capable of generating a highly purified neutrophil population
for further functional in-vitro assays. Consequently, this study
demonstrates a quick, cost effective, and easy-to-follow model, and may
be a significant alternative to isolation methods that need extra
subsequent steps such as flow cytometry-based cell sorting for reaching
highly purified neutrophil population.
Conclusion: The suggested combination of methods for the isolation and
cell migration analysis of human neutrophils is highly recommended to
use for disease models involving neutrophil migration such as
autoinflammatory disorders.
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