eISSN: 1644-4124
ISSN: 1426-3912
Central European Journal of Immunology
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SCImago Journal & Country Rank
3/2021
vol. 46
 
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abstract:
Letter to the Editor

Efficacy and specificity of different methods for human neutrophil extracellular trap isolation and handling

Fei Gao
1
,
Yun Guo
2

1.
Department of Intensive Care Unit, The Affiliated Wuxi People’s Hospital of Nanjing Medical University, China
2.
Department of Respiratory Medicine, The Affiliated Wuxi Children’s Hospital of Nanjing Medical University, China
Cent Eur J Immumol 2021; 46 (3): 384-387
Online publish date: 2021/09/28
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Introduction
Although in vitro incubation of various cell types with neutrophil extracellular traps (NETs) is commonly used to investigate the influence of NETs on cellular function, it is unclear which human NET isolation and handling protocol is superior. The present study sought to assess the efficacy (yield and purity) and efficiency (time taken) of different available human NET isolation and handling protocols.

Material and methods
Neutrophils isolated from human blood were stimulated using phorbol 12-myristate 13-acetate. Four distinct protocols were used to isolate NETs, and the yield was quantified using fluorimetry.

Results
Addition of the restriction enzyme AluI prior to centrifugation is unique to the most effective NET isolation method, yielding a NET concentration of 1077.22 ±229.04 ng/ml (at 523 nm) measured with PicoGreen. Immediate centrifugation to pellet neutrophils is unique to the most efficient method.

Conclusions
Balancing protocol efficacy and efficiency, the method incorporating centrifugation for 5 min at 450 × γ to pellet neutrophils is more than adequate.

keywords:

neutrophil, neutrophil extracellular traps, isolation, method comparison

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