One-step isolation of neutrophils using an elutriator

Abstract

To simplify the isolation of neutrophils, we developed a one-step procedure using elutriation. The perfusate (0.2% gelatin and 0.1% glucose in phosphate buffered saline) was pumped through an elutriator rotor at 4 ml/min (25 degrees C) with the rotor speed at 2370 rpm. Twenty milliliters of anticoagulated porcine venous blood were mixed with 60 ml of perfusate and loaded into the elutriator chamber. The flow rate was increased by 2 ml/min increments and 100-ml fractions of effluent were collected at each increment. Concentrations of neutrophils and mononuclear cells were measured in each fraction, and the percentage of total neutrophils or mononuclear cells was plotted against flow rate. The optimal yield (46%) and purity (95.1%) of neutrophils (n = 8) was obtained in pooled fractions at flow rates greater than 20 ml/min. Neutrophils in this preparation were round, the granules were intact, and the nuclei were lobulated. In addition, the cells produced superoxide in the presence of phorbol myristate acetate and phagocytosed zymosan particles. These characteristics were similar to those of porcine neutrophils prepared by a conventional sedimentation method. The yield (43%) and purity (94%) of human neutrophils isolated using the elutriator method was similar to that for porcine cells. This one-step method provides a moderate yield of pure neutrophils that have retained their morphology and function.