The density distribution of human mononuclear blood leukocytes was studied in order to define the optimal conditions for the separation of monocytes and lymphocytes by isopycnic centrifugation. Under standardized conditions, two populations of cells with partially overlapping, normally distributed densities were consistently found. The cells with the lowest density were recognized as monocytes, using phagocytosis and size distribution analysis as criteria. Since the density of monocytes continuously increased during the centrifugation, optimal separation of monocytes and lymphocytes could only be achieved by limiting the time of centrifugation to 10 min at 2200 g and 4 degrees C. The separation on discontinuous density gradients decreased when the load exceeded 8 X 10(6) mononuclear cells per sq cm. Analysis of the composition of the two cell populations obtained after separation on a three-layer discontinuous gradient revealed that the contamination of the monocytes with lymphocytes was due to the partial overlapping density distributions of both cell types. A small and a large scale method for isolation of monocytes from blood on discontinuous density gradients are presented. Under the described conditions, a preparation of functionally intact monocytes can be obtained which is comparable, both in yield and purity, to those obtained by methods based on surface adherence without the drawbacks of the latter methods.
During a study on the separation of human blood monocytes from lymphocytes, a method was developed to recognize and count monocytes by electronic means. Lightscattering (Cytograf, Bio/Physics), and changes in electrical resistance (Channelyzer, Coulter) were used to size mononuclear leukocytes directly in cell suspensions. Both methods revealed a size distribution profile in which two populations of mononuclear leukocytes could be distinguished. The largest cells were virtually eliminated after phagocytosis of iron particles. We confirmed that these cells were monocytes by three different criteria: the intracellular lysozyme activity, the number of phagocytes, and the percentage of cells with kidney-shaped nuclei. The highly significant correlations we found showed that monocytes could be recognized and counted by electronic sizing. For this method, purified mononuclear leukocyte preparations had to be used, since the presence of erythrocytes, platelets, and polymorphonuclear cells interfered. Statistical analysis revealed that electronic sizing permitted discrimination of differences in monocyte content of 4.5%, with a probability of 95%. It was calculated that this sensitivity of electronic monocyte counting was about three times higher than the sensitivity of microscopic methods. Since 100,000 cells can be sized within a few seconds, not only the efficiency of the preparation but also minor changes in the size of monocytes and lymphocytes introduced during the isolation can be followed.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.