Development of a simple method for rapid isolation of polymorphonuclear leukocytes from human blood.
Polymorphonuclear leukocytes (PMNs; commonly known as neutrophils) play essential roles in innate immunity and inflammation. Although there are standardized methods for the isolation of human neutrophils, they are time consuming and demand considerable technical expertise, making them unfeasible for many clinical applications. Here, we describe a simple and time-efficient technique for the isolation of human neutrophils, which adapts a readily available commercial cell preparation tube (CPT) currently in use for isolation of peripheral blood mononuclear cells (PBMC) and plasma and is now adapted to also yield neutrophils. The total time required for neutrophil isolation was less than 1 hr. Neutrophils isolated by this method were highly purified (> or =97%) as assessed by surface expression of the neutrophil specific marker, CD66b. Neutrophils isolated by this method were functional as demonstrated by their ability to secrete interleukin-1 receptor antagonist (IL-1RA). Neutrophils isolated using this new technique secreted significant amounts of soluble IL-1RA (929.3+/-197 pg/10(6)cells/mL) in response to lipopolysaccharide (LPS). Use of this adapted CPT method allows simultaneous isolation of functional human neutrophils as well as PBMC and plasma. Adoption of this new method will allow the conduct of different neutrophil assays at any clinical site without requiring trained laboratory personnel or a large staff time commitment.
De
Asit K
AK
Department of Surgery, University of Rochester Medical Center, Rochester, NY 14642, USA. asit_de@urmc.rochester.edu
Roach
Sarah E
SE
De
Mita
M
Minielly
Robin C
RC
Laudanski
Krzysztof
K
Miller-Graziano
Carol L
CL
Bankey
Paul E
PE
D015703:Antigens, CD / Q000032:analysis; D000943:Antigens, Differentiation / Q000032:analysis; D015815:Cell Adhesion Molecules; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured; D005434:Flow Cytometry; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein; D009504:Neutrophils / Q000166:cytology* / Q000378:metabolism; D012795:Sialoglycoproteins / Q000378:metabolism; D013997:Time Factors
D015703:Antigens, CD; D000943:Antigens, Differentiation; C069847:CD66 antigens; D015815:Cell Adhesion Molecules; C507763:IL1RN protein, human; D053590:Interleukin 1 Receptor Antagonist Protein; D012795:Sialoglycoproteins