A Nycodenz gradient method for the purification of neutrophils from the peripheral blood of rats

Abstract

A Nycodenz gradient technique is described which permits the separation of functionally active polymorphonuclear neutrophils (PMN) from the peripheral blood of rats. PMN are obtained at greater than 90% purity and fractionate at a peak density of 1.0919 g/ml. The method is suitable for isolating PMN when the circulating PMN count is low (less than 20%) as in normal rats and when the count is high (greater than 30%), as a result of inducing inflammation in the subcutaneous air pouch of rats by the intra-pouch injection of peptone. The chemotactic responsiveness of rat PMN was found to be markedly less than that of human PMN when the formyl peptides were used as chemoattractants but not when zymosan-activated serum was the chemoattractant. Blood PMN from normal rats and from peptone-treated rats showed no significant difference in their response to ZAS indicating that priming of their activity by the induction of long term (8 day) inflammation was not a feature of these experiments. However, air pouch-derived PMN displayed a highly significant reduction in activity compared with their isologous blood-PMN. The Nycodenz method offers an alternative to the Percoll separation method for rat blood and will be useful when comparative studies of elicited and non-elicited PMN are required since the latter are obtained in sufficiently high yield and purity for microassays on their function to be performed.