Cotter Matthew J, Muruve Daniel A
Department of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1.
J Immunol Methods. 2006 May 30;312(1-2):68-78. doi: 10.1016/j.jim.2006.02.019. Epub 2006 Apr 19.
Neutrophils are phagocytic leukocytes that represent one of the first lines of defense during infection and injury. Neutrophils emigrate into tissues during inflammation and are phenotypically different compared to cells in the circulation. To further understand the biology of tissue-recruited neutrophils, we have developed a reliable method to isolate these cells from inflamed liver. Acute liver inflammation was induced in mice by systemic treatment with adenovirus vectors. Two hours following adenovirus treatment, livers were enzymatically digested and leukocytes isolated by Percoll density gradient centrifugation. Neutrophils were then purified by negative immunomagnetic separation. Neutrophils isolated in this manner were 95% pure as determined by flow cytometry and more than 97% viable by propidium iodide staining. In order to carry out molecular studies, we extracted high quality genomic DNA and RNA from isolated neutrophils. PCR was used to successfully amplify sample genes from isolated neutrophil DNA. Isolated neutrophil RNA was used in a ribonuclease protection assay to evaluate chemokine gene expression. Neutrophils were shown to express multiple chemokine mRNA transcripts including MIP-1 beta, MIP-2 and IP-10. This work describes a novel method to isolate highly pure, viable neutrophils from pathologically inflamed tissue for subsequent detailed cellular and molecular analysis.
中性粒细胞是吞噬性白细胞，是感染和损伤时的第一道防线之一。中性粒细胞在炎症期间迁移到组织中，其表型与循环中的细胞不同。为了进一步了解组织募集的中性粒细胞的生物学特性，我们开发了一种可靠的方法，从炎症肝脏中分离这些细胞。通过用腺病毒载体进行全身治疗，在小鼠中诱导急性肝脏炎症。腺病毒治疗两小时后，对肝脏进行酶消化，并通过Percoll密度梯度离心分离白细胞。然后通过阴性免疫磁分离纯化中性粒细胞。通过流式细胞术测定，以这种方式分离的中性粒细胞纯度为95%，通过碘化丙啶染色，其活力超过97%。为了进行分子研究，我们从分离的中性粒细胞中提取了高质量的基因组DNA和RNA。PCR用于成功地从分离的中性粒细胞DNA中扩增样本基因。分离的中性粒细胞RNA用于核糖核酸酶保护试验，以评估趋化因子基因表达。结果显示中性粒细胞表达多种趋化因子mRNA转录本，包括MIP-1β、MIP-2和IP-10。这项工作描述了一种从病理炎症组织中分离高度纯净、有活力的中性粒细胞的新方法，用于后续详细的细胞和分子分析。
