Journal of Experimental Medicine, Vol 154, 1500-1516, Copyright © 1981 by Rockefeller University Press
Long-term culture and cloning of nontransformed human B lymphocytes
B Sredni, DG Sieckmann, S Kumagai, S House, I Green and WE Paul
B lymphocyte-enriched cell populations cultured with mitogens in initial
suspension cultures formed colonies in soft agar when the same mitogenic
agent was present in the lower layer of a two-layer soft agar system.
Colony formation depended upon the presence of T cells in the initial
culture, and was optimal after an initial 72-h culture with
phytohemagglutinin (PHA; 12.5 microliters/ml), pokeweed mitogen (PWM; 2.5
micrograms/ml), or protein A (10 micrograms/ml). The colonies could be
picked from the agar and propagated by feeding every 3 d with medium
supplemented with a growth factor-containing tissue culture supernate. The
growth factor-containing supernate was prepared by stimulating pools of
human peripheral blood mononuclear cells for 72 h with PHA or PWM. The
lines propagated in this manner were membrane Ig+, lacked sheep erythrocyte
rosette-forming ability, and did not ingest latex. They lacked the
Epstein-Barr nuclear antigen (EBNA) and had 46 chromosomes. Such lines have
been propagated for over 1 yr. One line (BL1) was subjected to limiting
dilution cloning and a line, BL1.1, was prepared that contained 96%
lambda-bearing cells and no kappa-bearing cells. This line was also EBNA
negative. This procedure can thus be used to prepare and clone long-term
lines of nontransformed human B lymphocytes.
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