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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: 2.66±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.