Isolation of neutrophils from mouse liver: A novel method to study effector leukocytes during inflammation.
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.
Cotter
Matthew J
MJ
Department of Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1.
Muruve
Daniel A
DA
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D039481:CD11b Antigen / Q000032:analysis; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D018925:Chemokines / Q000235:genetics / Q000378:metabolism; D002634:Chemotaxis, Leukocyte; D004247:DNA / Q000032:analysis; D005822:Genetic Vectors / Q000235:genetics; D006505:Hepatitis / Q000276:immunology*; D007249:Inflammation; D007962:Leukocytes / Q000276:immunology; D008099:Liver / Q000276:immunology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009504:Neutrophils / Q000276:immunology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D014158:Transcription, Genetic
D039481:CD11b Antigen; D018925:Chemokines; D012333:RNA, Messenger; D004247:DNA