The separation of different cell classes from lymphoid organs. VI. The effect of osmolarity of gradient media on the density distribution of cells.

Ficoll, a polymer of sucrose, and bovine serum albumin have been compared as media for preparation of density gradients for separation of lymphoid cells. Direct vapour pressure osmometer measurements demonstrated that Ficoll at high concentrations has a marked effect on the osmolarity of the medium in which it is dissolved. Albumin, prepared in the manner we have described in earlier papers of this series, has no effect, and density gradients prepared with this albumin do not contain gradients of osmolarity. Antibody forming cells give basically similar density distributions after separation in both media, apart from a slight overall shift to the dense end due to shrinkage in Ficoll. Heterogeneous, multiple peak distribution profiles, or relatively simple, homogeneous profiles may be obtained in both media, depending on the source of antibody-forming cells, the stage of differentiation of the population, and the degree of resolution of the gradient. Antibody-forming cell density heterogeneity is a property of the cell population, rather than an artifact of the separation system. Different animal species have a wide variation in the osmolarity of their sera. Albumin-gradients of osmolarity which were not iso-osmotic for the mouse caused a general shift in the density of mouse cell populations but no marked change in the relative distribution of cells. However, a change from iso-osmotic conditions did affect the ability of cells to reband on a second gradient. The use of gradients iso-osmotic for the particular species under study is recommended as a general procedure.