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Use of Size Fractionation of in Vitro‐Activated Human B Lymphocytes for Studies of Cell Cycle‐Dependent Growth Regulation
Author(s) -
ERIKSTEIN B. K.,
SCHWARZE P.,
BLOMHOFF H. KIIL,
FUNDERUD S.,
STOKKE T.,
SMELAND E. B.
Publication year - 1991
Publication title -
scandinavian journal of immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.934
H-Index - 88
eISSN - 1365-3083
pISSN - 0300-9475
DOI - 10.1111/j.1365-3083.1991.tb02491.x
Subject(s) - thymidine , elutriation , in vitro , dna synthesis , incubation , biology , microbiology and biotechnology , cell cycle , cell growth , fractionation , cell , chemistry , biochemistry , chromatography , organic chemistry
Cell cycle progression of in vitro‐stimulated human B lymphocytes occurs a synchronously. In order lo allow detailed studies of growth Control G 1 . B cells Were stimulated with anti‐μ and low molecular weight B‐cell growth factor (LMW BCGF) for 50 h and subsequently separated into nine fractions of cells by means of centrifugal elutriation. As judged by volume profiles, activation antigen expression and DNA content, the cells in fractions t 4 were in early Io mid‐G 1 , while fractions 5 7 mainly contained cells in late G 1 . and fractions 8 9 contained cells mainly in Sand G 2 , Cells in fractions 5–7 had passed the commitment point, as demonstrated by a high spontaneous incorporation of [ 3 H]thymidine when recultured in medium alone. Moreover S‐phase entry of these cells was largely unaffected by exogenous growth‐promoting or growth‐inhibitory signals. Cells in early (fractions 1 2) and intermediate fractions (fractions 3 4) showed a negligible spontaneous 3 [H]thymidine incorporation, but a significant proportion of these cells progressed to S phase upon restimulation. Moreover, while IL‐4 or the anti‐CD40 MoAb G28‐ 5 potently stimulated cells in early and intermediate fractions, the responsiveness to LMW BCGF alone was obtained Just prior to the commitment point.