Improved methods for separating hydrolytic granules of neutrophil leucocytes.

Abstract

Highly efficient methods for isolating two hydrolytic granules of neutrophils are described. Neutrophil obtained from guinea pig peritoneal exudate cells were washed extensively with isotonic sucrose and then treated with heparin. More than 95 per cent of the cells so treated were disrupted with a Dounce homogenizer. Since nuclei were broken, leaving other organelles intact, homogenates were incubated with DNase to reduce viscosity. Postnuclear supernatants were centrifuged on a discontinuous gradient of Percoll. Azurophil granules, high in beta-glucuronidase activity, sedimented at fractions of d = 1.081 and showed very little activity of other marker enzymes. High neutral alpha-glucosidase activity was observed in granular fractions of d = 1.038 and it is suggested that this is a marker for specific granules of neutrophils.