Cellular activity of murine phagocytes isolated from peripheral blood by a discontinuous gradient

A mixture of Ficoll 400 and sodium diatrizoate (Hypaque) at a density of 1.077 g/ml has been used to isolate the mononuclear cells from the remaining haematic cells. A simple, inexpensive and classical method was established to obtain substantially erythrocyte-free polymorphonuclear cell preparations from mouse peripheral blood, using a mixture of the same substances but at a density of 1.119 g/ml. This method along with that at a density of 1.077 g/ml allows two cellular bands to appear which contain mononuclear and polymorphonuclear (PMN) cells, respectively. Using this method, the counts of monocytes isolated from peripheral blood are significantly greater than those obtained by a one-step Ficoll-Hypaque procedure. On the contrary, the counts of PMN cells are significantly smaller than when sedimentation in dextran (6% solution) is used after gradient centrifugation. In this paper, chemiluminescence assay has been used to analyze the possible variations in phagocytic activity of cells isolated by both procedures, since it appears to be one of the most sensitive methods available for this purpose. The results obtained show a slightly greater activation in monocytes and PMN cells isolated by one-step Ficoll-Hypaque procedure, in comparison with another method which uses both Ficoll-Hypaque 1077 and Ficoll-Hypaque 1119, although statistical differences were not significant.