Optimization of methods for the accurate characterization of whole blood neutrophils.
Optimization of methods for the accurate characterization of whole blood neutrophils.
复制标题
优化方法，以准确表征全血中性粒细胞。
DOI：
10.1038/s41598-022-07455-2
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发表时间：
2022-03-07
影响因子：
4.6
通讯作者：
Hel Z
中科院分区：
文献类型：
--
作者：
Connelly AN;Huijbregts RPH;Pal HC;Kuznetsova V;Davis MD;Ong KL;Fay CX;Greene ME;Overton ET;Hel Z
中性粒细胞是最丰富的循环白细胞群体，在免疫防御、先天性和适应性免疫系统的调节以及疾病发病机制中具有关键作用。我们在理解中性粒细胞活化、募集和功能的精确机制方面的进展受到了缺乏用于这种容易活化的群体的表征和表型分析的优化和标准化方法的阻碍。通过比较八种中性粒细胞表征方法，我们证明了中性粒细胞活化和脱粒的水平与特定的实验条件以及所采用的操作步骤的数量和类型相关。在一步固定和红细胞裂解之前，在4 °C下对全血进行染色并去除剩余的未结合抗体，可最大限度地减少中性粒细胞活化，减少处理过程中的表型改变，并防止非特异性抗体结合。用于收集的抗凝剂，处理延迟，以及样本分析过程中的时间和温度对中性粒细胞表型的影响。所提供的数据为中性粒细胞表征的更高质量标准提供了基础，提高了研究的一致性和重现性。
Neutrophils are the most abundant circulating leukocyte population with critical roles in immune defense, regulation of innate and adaptive immune systems, and disease pathogenesis. Our progress in understanding precise mechanisms of neutrophil activation, recruitment, and function has been hampered by the lack of optimized and standardized methods for the characterization and phenotyping of this readily activated population. By comparing eight methods of neutrophil characterization, we demonstrate that the level of neutrophil activation and degranulation is associated with specific experimental conditions and the number and type of manipulation steps employed. Staining whole blood at 4 °C and removal of remaining unbound antibodies prior to one-step fixation and red blood cell lysis minimizes neutrophil activation, decreases phenotypic alterations during processing, and prevents nonspecific antibody binding. The effects of anticoagulants used for collection, processing delays, and time and temperature during sample analysis on neutrophil phenotype are addressed. The presented data provide a foundation for higher quality standards of neutrophil characterization improving consistency and reproducibility among studies.
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影响因子：
7.3
作者：
Brown, RA;Lever, R;Page, CP
通讯作者：
Page, CP
影响因子：
7.3
作者：
Pocock JM;Storisteanu DML;Reeves MB;Juss JK;Wills MR;Cowburn AS;Chilvers ER
通讯作者：
Chilvers ER
影响因子：
32.4
作者：
Adrover, Jose M.;del Fresno, Carlos;Hidalgo, Andres
通讯作者：
Hidalgo, Andres
影响因子：
15.9
作者：
Pillay, Janesh;Kamp, Vera M.;Koenderman, Leo
通讯作者：
Koenderman, Leo
影响因子：
6.7
作者：
Bowers NL;Helton ES;Huijbregts RP;Goepfert PA;Heath SL;Hel Z
通讯作者：
Hel Z