Thomas Huw B, Moots Robert J, Edwards Steven W, Wright Helen L
Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.
Institute of Ageing and Chronic Disease, University Hospital Aintree, University of Liverpool, Liverpool, United Kingdom.
PLoS One. 2015 Sep 24;10(9):e0138982. doi: 10.1371/journal.pone.0138982. eCollection 2015.
Protocols for the isolation of neutrophils from whole blood often result in neutrophil preparations containing low numbers (~5%) of contaminating leukocytes, and it is possible that these contaminating cells contribute to highly sensitive assays that measure neutrophil gene expression (e.g. qPCR). We investigated the contribution of contaminating leukocytes on the transcriptome profile of human neutrophils following stimulation with inflammatory cytokines (GM-CSF, TNFα), using RNA-Seq. Neutrophils were isolated using Polymorphprep or the StemCell untouched neutrophil isolation kit (negative selection of "highly pure" neutrophils). The level of contamination was assessed by morphology and flow cytometry. The major source of contamination in Polymorphprep neutrophil preparations was from eosinophils and was highly donor dependent. Contaminating cells were largely, but not completely, absent in neutrophil suspensions prepared using negative selection, but the overall yield of neutrophils was decreased by around 50%. RNA-seq analysis identified only 25 genes that were significantly differentially-expressed between Polymorphprep and negatively-selected neutrophils across all three treatment groups (untreated, GM-CSF, TNFα). The expression levels of 34 cytokines/chemokines both before and after GM-CSF or TNFα treatment were not significantly different between neutrophil isolation methods and therefore not affected by contributions from non-neutrophil cell types. This work demonstrates that low numbers (<5%) of contaminating leukocytes in neutrophil preparations contribute very little to the overall gene expression profile of cytokine-stimulated neutrophils, and that protocols for the isolation of highly pure neutrophils result in significantly lower yields of cells which may hinder investigations where large numbers of cells are required or where volumes of blood are limited.
从全血中分离中性粒细胞的方案通常会导致中性粒细胞制剂中含有少量（约5%）的污染白细胞，并且这些污染细胞有可能会对测量中性粒细胞基因表达的高灵敏度检测方法（如定量PCR）产生影响。我们使用RNA测序技术，研究了炎症细胞因子（GM-CSF、TNFα）刺激后，污染白细胞对人中性粒细胞转录组图谱的影响。使用Polymorphprep或StemCell未触及中性粒细胞分离试剂盒（阴性选择“高纯度”中性粒细胞）分离中性粒细胞。通过形态学和流式细胞术评估污染水平。Polymorphprep中性粒细胞制剂中的主要污染源来自嗜酸性粒细胞，且高度依赖供体。在使用阴性选择制备的中性粒细胞悬液中，污染细胞大部分但并非完全不存在，不过中性粒细胞的总体产量下降了约50%。RNA测序分析仅鉴定出在所有三个治疗组（未处理、GM-CSF、TNFα）中，Polymorphprep和阴性选择的中性粒细胞之间有25个基因存在显著差异表达。在GM-CSF或TNFα处理前后，34种细胞因子/趋化因子的表达水平在中性粒细胞分离方法之间没有显著差异，因此不受非中性粒细胞类型的影响。这项研究表明，中性粒细胞制剂中少量（<5%）的污染白细胞对细胞因子刺激的中性粒细胞的整体基因表达谱贡献很小，并且分离高纯度中性粒细胞的方案会导致细胞产量显著降低，这可能会阻碍需要大量细胞或血液量有限的研究。
