Abstract
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.
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Selected References
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- Bach M. K., Brashler J. R. Isolation of subpopulations of lymphocytic cells by the use of isotonically balanced solutions of Ficoll. I. Development of methods and demonstration of the existence of a large but finite number of subpopulations. Exp Cell Res. 1970 Aug;61(2):387–396. doi: 10.1016/0014-4827(70)90462-3. [DOI] [PubMed] [Google Scholar]
- Cunningham A. J., Szenberg A. Further improvements in the plaque technique for detecting single antibody-forming cells. Immunology. 1968 Apr;14(4):599–600. [PMC free article] [PubMed] [Google Scholar]
- Diener E. A new method for the enumeration of single antibody-producing cells. J Immunol. 1968 May;100(5):1062–1070. [PubMed] [Google Scholar]
- Haskill J. S., Legge D. G., Shortman K. Density distribution analysis of cells forming 19S hemolytic antibody in the rat. J Immunol. 1969 Mar;102(3):703–712. [PubMed] [Google Scholar]
- Kraft N., Shortman K., Marchalonis J. Density distribution analysis of a primary antibody forming cell response. Immunology. 1971 Jun;20(6):919–930. [PMC free article] [PubMed] [Google Scholar]
- Legge D. G., Shortman K. The effect of pH on the volume, density and shape of erythrocytes and thymic lymphocytes. Br J Haematol. 1968 Mar;14(3):323–335. doi: 10.1111/j.1365-2141.1968.tb01503.x. [DOI] [PubMed] [Google Scholar]
- Niewisch H., Vogel H., Matioli G. Concentration, quantitation, and identification of hemopoietic stem cells. Proc Natl Acad Sci U S A. 1967 Dec;58(6):2261–2267. doi: 10.1073/pnas.58.6.2261. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shortman K. The density distribution of thymus, thoracic duct and spleen lymphocytes. J Cell Physiol. 1971 Jun;77(3):319–330. doi: 10.1002/jcp.1040770306. [DOI] [PubMed] [Google Scholar]
- Shortman K. The separation of different cell classes from lymphoid organs. II. The purification and analysis of lymphocyte populations by equilibrium density gradient centrifugation. Aust J Exp Biol Med Sci. 1968 Aug;46(4):375–396. doi: 10.1038/icb.1968.32. [DOI] [PubMed] [Google Scholar]
- van Dicke KA Hooft J. I., van Bekkum D. W. The selective elimination of immunologically competent cells from bone marrow and lymphatic cell mixtures. II. Mouse spleen cell fractionation on a discontinuous albumin gradient. Transplantation. 1968 Jul;6(4):562–570. doi: 10.1097/00007890-196807000-00009. [DOI] [PubMed] [Google Scholar]