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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: 2.66×106±0.22 PMNs per ml of blood (Method-A) and 1.87×106±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.