title: Reduced ammonium chloride haemolysis time enhances the number of isolated functional rabbit polymorphonuclear neutrophils.
abstract: In the present study we compared seven different methods for isolating rabbit polymorphonuclear neutrophils (PMNs) with a view to assessing viability, lymphocyte contamination and isolation yield. The two methods offering the best isolation yield and functional PMNs were retained. Leukocyte-containing plasma fraction was obtained after erythrocyte sedimentation with dextran. First, PMNs were isolated from this fraction, using hypotonic ammonium chloride haemolysis followed by Histopaque density gradient centrifugation (Method-A). Second, PMNs were obtained from leukocyte-containing plasma after centrifugation on two Percoll layers (Method-B). These processes resulted in a high cellular yield: 2.66x10(6)+/-0.22 PMNs per ml of blood (Method-A) and 1.87x10(6)+/-0.37 PMNs per ml of blood (Method-B). In both cases the PMNs isolated were of high purity and viability. In comparison, when using the standard techniques for rabbit - consisting of ammonium chloride haemolysis taking at least four times as long--fewer PMNs were isolated. The PMNs isolated by Method-A and -B were able to generate a high amount of reactive oxygen species (ROS) after stimulation with phorbol 12-myristate 13-acetate (PMA). These methods to separate PMNs are recommended for in vitro studies.
authors.affiliation: Faculté des Sciences Pharmaceutiques et Biologiques, Laboratoire de Pharmacologie, Pharmacocinétique et Pharmacie Clinique, Lille, France.
authors.forename: F
authors.initials: F
authors.lastname: Kouoh
authors.forename: H
authors.initials: H
authors.lastname: Levert
authors.forename: B
authors.initials: B
authors.lastname: Gressier
authors.forename: M
authors.initials: M
authors.lastname: Luyckx
authors.forename: C
authors.initials: C
authors.lastname: Brunet
authors.forename: T
authors.initials: T
authors.lastname: Dine
authors.forename: L
authors.initials: L
authors.lastname: Ballester
authors.forename: M
authors.initials: M
authors.lastname: Cazin
authors.forename: J C
authors.initials: JC
authors.lastname: Cazin
mesh_terms: D000643:Ammonium Chloride / Q000494:pharmacology*; D000818:Animals; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival; D006461:Hemolysis / Q000187:drug effects / Q000502:physiology*; D066298:In Vitro Techniques; D007958:Leukocyte Count; D008297:Male; D009504:Neutrophils / Q000166:cytology* / Q000502:physiology*; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000378:metabolism; D013481:Superoxides / Q000097:blood
chemical_list: D017382:Reactive Oxygen Species; D000643:Ammonium Chloride; D013481:Superoxides