Comparison of different methods for the purification of eosinophils from human peripheral blood

Eosinophils are often purified in discontinuous gradients. Since continuous gradients usually provide a greater recovery of more highly purified cells, the present investigation was undertaken to compare the purification of eosinophils from normal whole blood in continuous and discontinuous gradients of Percoll. Contrary to our expectations, recovery and purity of eosinophils obtained from the discontinuous gradients were comparable to or higher than those from the continuous gradients of Percoll that were tested with whole blood. The purity of the modal fractions of eosinophils from the discontinuous gradients was between 88 and greater than 99% of the nucleated cells and from the continuous gradients, 80 to 93% of the nucleated cells. We have compared continuous and discontinuous gradients with many different kinds of cells. This is the first time we have found continuous and discontinuous gradients equally effective. We speculate this finding is related to the fact that the band capacities are vastly overloaded in these gradients. In addition, we tested the rate of superoxide production by eosinophils from the same donors after their purification by two different methods in discontinuous gradients. Eosinophils purified from normal whole blood in gradients of Percoll by a modification of the method of Roberts and Gallin [1985) Blood 65, 433-440) had a higher rate of superoxide production after stimulation with phorbol myristate acetate than those purified from leukocyte-rich plasma in gradients of Metrizamide by the method of Vadas et al. [1979) J. Immunol. 122, 1228-1236).