Reduced ammonium chloride haemolysis time enhances the number of isolated functional rabbit polymorphonuclear neutrophils.
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.
Kouoh
F
F
Faculté des Sciences Pharmaceutiques et Biologiques, Laboratoire de Pharmacologie, Pharmacocinétique et Pharmacie Clinique, Lille, France.
Levert
H
H
Gressier
B
B
Luyckx
M
M
Brunet
C
C
Dine
T
T
Ballester
L
L
Cazin
M
M
Cazin
J C
JC
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
D017382:Reactive Oxygen Species; D000643:Ammonium Chloride; D013481:Superoxides