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. 1978 Feb 1;147(2):279–296. doi: 10.1084/jem.147.2.279

Mouse spleen lymphoblasts generated in vitro. Recovery in high yield and purity after floatation in dense bovine plasma albumin solutions

PMCID: PMC2184489  PMID: 564389

Abstract

Mouse spleen lymphoblasts, stimulated to divide in vitro, acquired a low cell density and could be separated by isopycnic techniques. Cultured cells were suspended in BPA columns, rho = 1.080, and spun to equilibrium. The method was simple, fast, accomodated large numbers of cells, and was reproducible. It provided lymphoblasts in high yield and purity (at least 80% of the low density cells were blasts). It allowed for the recovery of proliferating cells in their first cell cycle, and did not alter the subsequent ability of cells to proliferate when recultured in vitro. Certain properties of mouse spleen lymphoblasts were analyzed in detail. Lymphoblasts induced by LPS, FCS, con A (tetravalent and succinylated), and MLC were very similar except in the absolute numbers that were induced. The blasts exhibited the classic cytologic features of enlarged nucleoli and abundant cytoplasmic polyribosomes (basophilia). As a population, they were enlarged in size relative to nondividing cells, but this seemed to apply primarily to cells in the S and G2+ M phase of the cell cycle rather than G1. The cell cycle distribution of lymphoblasts was analyzed by flow microfluorometry. By analyzing low density cells obtained at varying intervals after mitogen stimulation, FMF indicated that lymphoblasts enter the S phase of their first cell cycle beginning at 20-24 h after stimulation.

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Selected References

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  1. Andersson J., Möller G., Sjöberg O. Selective induction of DNA synthesis in T and B lymphocytes. Cell Immunol. 1972 Aug;4(4):381–393. doi: 10.1016/0008-8749(72)90040-8. [DOI] [PubMed] [Google Scholar]
  2. Andersson L. C., Häyry P. Clonal isolation of alloantigen-reactive T-cells and characterization of their memory functions. Transplant Rev. 1975;25:121–162. doi: 10.1111/j.1600-065x.1975.tb00728.x. [DOI] [PubMed] [Google Scholar]
  3. Andersson L. C., Nordling S., Häyry P. Proliferation of B and T cells in mixed lymphocyte cultures. J Exp Med. 1973 Jul 1;138(1):324–329. doi: 10.1084/jem.138.1.324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Basham T. Y., Waxdal M. J. Cross-linking of T cell mitogens: effects on B and T cell proliferation and immunoglobulin synthesis. J Immunol. 1977 Mar;118(3):863–868. [PubMed] [Google Scholar]
  5. Bowers W. E. Lysosomes in rat thoracic duct lymphocytes fractionated by zonal centrifugation. J Cell Biol. 1973 Oct;59(1):177–184. doi: 10.1083/jcb.59.1.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Douglas S. D. Human lymphocyte growth in vitro: morphologic, biochemical, and immunologic significance. Int Rev Exp Pathol. 1971;10:41–114. [PubMed] [Google Scholar]
  7. Everson L. K., Buell D. N., Rogentine G. N., Jr Separation of human lymphoid cells into G 1 , S, and G 2 cell cycle populations by use of a velocity sedimentation technique. J Exp Med. 1973 Feb 1;137(2):343–358. doi: 10.1084/jem.137.2.343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fried J. Analysis of deoxyribonucleic acid histograms from flow cytofluorometry. Estimation of the distribution of cells within S phase. J Histochem Cytochem. 1977 Jul;25(7):942–951. doi: 10.1177/25.7.894010. [DOI] [PubMed] [Google Scholar]
  9. Fried J. Method for the quantitative evaluation of data from flow microfluorometry. Comput Biomed Res. 1976 Jun;9(3):263–276. doi: 10.1016/0010-4809(76)90006-9. [DOI] [PubMed] [Google Scholar]
  10. Fried J., Perez A. G., Clarkson B. D. Flow cytofluorometric analysis of cell cycle distributions using propidium iodide. Properties of the method and mathematical analysis of the data. J Cell Biol. 1976 Oct;71(1):172–181. doi: 10.1083/jcb.71.1.172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fried J., Yataganas X., Kitahara T., Perez A., Ferguson R., Sullivan S., Clarkson B. Quantitative analysis of flow microfluorometric data from asynchronous and drug-treated cell populations. Comput Biomed Res. 1976 Jun;9(3):277–290. doi: 10.1016/0010-4809(76)90007-0. [DOI] [PubMed] [Google Scholar]
  12. GOLDSTEIN I. J., HOLLERMAN C. E., SMITH E. E. PROTEIN-CARBOHYDRATE INTERACTION. II. INHIBITION STUDIES ON THE INTERACTION OF CONCANAVALIN A WITH POLYSACCHARIDES. Biochemistry. 1965 May;4:876–883. doi: 10.1021/bi00881a013. [DOI] [PubMed] [Google Scholar]
  13. GOWANS J. L. The fate of parental strain small lymphocytes in F1 hybrid rats. Ann N Y Acad Sci. 1962 Oct 24;99:432–455. doi: 10.1111/j.1749-6632.1962.tb45326.x. [DOI] [PubMed] [Google Scholar]
  14. Graham R. C., Jr, Karnovsky M. J. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem. 1966 Apr;14(4):291–302. doi: 10.1177/14.4.291. [DOI] [PubMed] [Google Scholar]
  15. Gunther G. R., Wang J. L., Edelman G. M. The kinetics of cellular commitment during stimulation of lymphocytes by lectins. J Cell Biol. 1974 Aug;62(2):366–377. doi: 10.1083/jcb.62.2.366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gunther G. R., Wang J. L., Yahara I., Cunningham B. A., Edelman G. M. Concanavalin A derivatives with altered biological activities. Proc Natl Acad Sci U S A. 1973 Apr;70(4):1012–1016. doi: 10.1073/pnas.70.4.1012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Janossy G., Shohat M., Greaves M. F., Dourmashkin R. R. Lymphocyte activation. IV. The ultrastructural pattern of the response of mouse T and B cells to mitogenic stimulation in vitro. Immunology. 1973 Feb;24(2):211–227. [PMC free article] [PubMed] [Google Scholar]
  18. Klaus G. G., Janossy G., Humphrey J. H. The immunological properties of haptens coupled to thymus-independent carrier molecules. III. The role of the immunogenicity and mitogenicity of the carrier in the induction of primary IgM anti-hapten responses. Eur J Immunol. 1975 Feb;5(2):105–111. doi: 10.1002/eji.1830050207. [DOI] [PubMed] [Google Scholar]
  19. Krishan A. Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Biol. 1975 Jul;66(1):188–193. doi: 10.1083/jcb.66.1.188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lohrmann H. P., Graw C. M., Graw R. G., Jr Stimulated lymphocyte cultures: responder recruitment and cell cycle kinetics. J Exp Med. 1974 May 1;139(5):1037–1048. doi: 10.1084/jem.139.5.1037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Loos J. A., Roos D. Isopycnic centrifugation of antigen-activated human blood lymphocytes in the early phase of in vitro stimulation. Exp Cell Res. 1974 Jun;86(2):342–350. doi: 10.1016/0014-4827(74)90722-8. [DOI] [PubMed] [Google Scholar]
  22. MacDonald H. R., Phillips R. A., Miller R. G. Allograft immunity in the mouse. II. Physical studies of the development of cytotoxic effector cells from their immediate progenitors. J Immunol. 1973 Aug;111(2):575–589. [PubMed] [Google Scholar]
  23. Macdonald H. R., Miller R. G. Synchronization of mouse L-cells by a velocity sedimentation technique. Biophys J. 1970 Sep;10(9):834–842. doi: 10.1016/S0006-3495(70)86338-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. PEARMAIN G., LYCETTE R. R., FITZGERALD P. H. Tuberculin-induced mitosis in peripheral blood leucocytes. Lancet. 1963 Mar 23;1(7282):637–638. doi: 10.1016/s0140-6736(63)91275-3. [DOI] [PubMed] [Google Scholar]
  25. ROBBINS J. H. TISSUE CULTURE STUDIES OF THE HUMAN LYMPHOCYTE. Science. 1964 Dec 25;146(3652):1648–1654. doi: 10.1126/science.146.3652.1648. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Splinter T. A., Reiss M. Separation of lymphoid-line cells according to volume and density. Exp Cell Res. 1974 Dec;89(2):343–351. doi: 10.1016/0014-4827(74)90799-x. [DOI] [PubMed] [Google Scholar]
  28. Steinman R. M., Cohn Z. A. Identification of a novel cell type in peripheral lymphoid organs of mice. I. Morphology, quantitation, tissue distribution. J Exp Med. 1973 May 1;137(5):1142–1162. doi: 10.1084/jem.137.5.1142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Steinman R. M., Cohn Z. A. Identification of a novel cell type in peripheral lymphoid organs of mice. II. Functional properties in vitro. J Exp Med. 1974 Feb 1;139(2):380–397. doi: 10.1084/jem.139.2.380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sörén L. Variability of the time at which PHA-stimulated lymphocytes initiate DNA synthesis. Exp Cell Res. 1973 Mar 30;78(1):201–208. doi: 10.1016/0014-4827(73)90055-4. [DOI] [PubMed] [Google Scholar]
  31. Wilson D. B., Blyth JL NOWELL P. C. Quantitative studies on the mixed lymphocyte interaction in rats. 3. Kinetics of the response. J Exp Med. 1968 Nov 1;128(5):1157–1181. doi: 10.1084/jem.128.5.1157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Zimmerman D. H., Kern M. Differentiation of lymphoid cells: effect of serum and other mitogenic agents on the selective induction of immunoglobulin M secreting lymph node cells in tissue culture. J Immunol. 1973 Nov;111(5):1326–1333. [PubMed] [Google Scholar]

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