A simple method for isolating human and rabbit polymorphonuclear neutrophils (PMNs)

Abstract

We report the successful application to human venous blood of a novel method developed to purify rabbit polymorphonuclear neutrophils (PMNs) from whole blood. Human PMNs were separated from whole blood after sedimentation with dextran and histopaque density gradient centrifugation. 3.92±0.26 x 106 PMNs per ml of blood was harvested. The purity of the preparation was 92.00±1.10%. The PMNs isolated were capable of generating a high amount of reactive oxygen species (ROS) and elastase after stimulation with phorbol 12-myristate 13-acetate (PMA): 13.43±0.3 μM of O2-, 9.62±0.15 μM of H2O2 and 5.48±0.01 μM of elastase. This method gives equivalent yield and viability when applied to isolating human or rabbit PMNs, in comparison with standard methods used to isolate human PMNs. Our method could be usefully exploited for comparative studies of rabbit and human PMNs with a cellular model of inflammatory oxidative stress in which the monitoring parameters are ROS and elastase. Thus, the results of animal (rabbit) studies can be extended to human diseases.