Growth of B-lymphocytes clones in semisolid culture is mitogen dependent.

Abstract

In vivo cloning techniques have made it possible to estimate the frequency, triggering requirements, and proliferative potential of individual B lymphocytes, as well as the diversity of major class and specificity of their end products (1-3). Recently, an in vitro technique for cloning murine B lymphocytes in agar has been described (4). In this report, we present evidence that a polysaccharide B-cell mitogen/polyclonal activator is present in unrefined agar and it or another appropriate B-cell mitogen is essential to B-cell proliferation in semisolid cultures. Semisolid Cultures. Single spleen cell suspensions were prepared, freed of coarse debris by settling and washed once with RPMI 1640 medium containing 10% fetal calf serum. Single strength McCoy's modified 5a medium plus 15% fetal calf serum, 2 mM L-glutamine, 16 ~g/ml Lasparagine, 8 ~g/ml L-serine, and 5 x 10 -3 M 2-mercaptoethanol (2-ME) was warmed to 37°C and mixed with a 1/10 volume of boiled 3% Bacto agar in H20. Spleen cells (2 x 104) were added and 1-ml aliquots were allowed to gel in 35-mm tissue culture dishes. Alternatively, agarose at 0.2% final concentration or 1% methyl cellulose were used instead of agar. Granulocyte-macrophage progenitors (CFU-c) were stimulated with an optimal concentration of medium conditioned by murine myelomonocytic leukemia (WEHI 3) cells in cultures of 7.5 x 104 bone marrow cells using the same medium without mercaptoethanol. Cultures were held at 37°C in a humidified atmosphere of 10% CO2-90% air for 5-7 days before examination with a dissecting microscope. Materials and MethodsLiquid Cultures. Duplicate cultures of spleen cells (2 x 10~/ml) were prepared in RPMI 1640 medium containing 5% fetal calf serum and 5 x 10 -5 M 2-ME in 0.2-ml volumes plus 0.01 ml of mitogen or phosphate-buffered saline (PBSL After 3 days incubation at 37°C and 10% CO2, the cultures were labeled by the addition of 0.2 ~Ci/well of '2~I-iododeoxyuridine ('25IUdR) (200 Ci/ mmol; New England Nuclear, Boston, Mass.). After an additional 3 h at 37°C, cultures were harvested on filter paper, washed with TCA using a semiautomatic cell harvester, and counted in a gamma counter. Lipopolysaccharide from Salmonella thyphosa WO901 (Difco Laboratories, Detroit, Mich.) and dextran sulfate (500,000 mol wt; Sigma Chemical Co., St. Louis, Mo.) were used as mitogens.Extraction and Chemical Characterization of Agar Mitogen. Bacto agar Clot no. 504388 and 551975; Difco Laboratories) was extracted with 37°C H20 for 1 h with occasional shaking, filtered through Whatman no. 1 paper on a scintered glass funnel, and lyophilized. Up to 80 mg was recovered/g of agar. Before use the material was dissolved in 37°C H20 and subjected to one of the *

Regulation of B-lymphocyte clonal proliferation by stimulatory and inhibitory macrophage-derived factors

“…Responses in liquid cultures were assessed after 72 h, whereas focal B-cell proliferation in semisolid medium is optimally observed after 6 days of culture. Colony formation is dependent upon 2-ME (16), mitogens native to laboratory grade agar (17) and serum, whereas B-cell activation by LPS in liquid cultures is demonstrable in the absence of 2-ME and serum (P. W. Kincade, unpublished observations). B-cell clonal expansion in soft agar medium is probably more fastidious than short-term liquid culture responses and the semisolid colony assay may be uniquely suited to studying the role of accessory cells because macrophage-lymphocyte, as well as lymphocyte-lymphocyte contact is prevented.…”

“…Colony formation is mitogen dependent (17) and the addition of the B-cell mitogen LPS, or SRBC enhances colony formation, particularly when few B cells are cultured (20). We therefore compared the dose response of lymph node cells cultured with or without these agents.…”

“…After 72 h of incubation at 37°C in 7% CO~ in humidified air, 0.2/xCi [l=I]iododeoxyuridine ([~2sI]IUDR; sp act 200 Ci/mM, New England Nuclear, Boston, Mass.) was added to the culture wells for an additional 3 h. The cells were harvested by a semi-automatic cell harvester, deposited on filter paper, washed with saline and 10% trichloroacetic acid (TCA), and the incorporation of [~2sI]IUDR counted in a gamma counter, as previously described (17). quirement for macrophages in B-cell colony formation and the ability of our two-layer culture system to satisfy this requirement was initially tested by using B lymphocytes from various tissue sources (Table I).…”

TEL-AML1 transgenic zebrafish model of precursor B cell acute lymphoblastic leukemia

“…Total cell counts showed a modest decrease in myeloid, and a slight increase of erythroid cells associated with the progenitor cell expansion. To assess the lymphoid proliferative potential of these precursors, kidney marrow progenitors were subjected to an in vitro mitogen-induced B cell clonogenic assay (29) (Fig. 2B and Table 1).…”

Deregulated Bcl-2-immunoglobulin transgene expands a resting but responsive immunoglobulin M and D-expressing B-cell population.

“…Primary spleen cell cultures were established with Dulbecco modified Eagle medium supplemented with 5% fetal calf serum as described previously (19). Spleen cell suspensions for the B-cell colony (CFU-B) assay were prepared at a density of 2.5 x 105 mononuclear cells per ml in McCoy medium (GIBCO Laboratories, Grand Island, N.Y.) supplemented with 15% fetal calf serum (Hyclone Laboratories, Logan, Utah), 2 mM L-glutamine, 16 ,ug of L-asparagine per ml, 8 ,ug of L-serine per ml, and 5 x 10-' M beta-mercaptoethanol, similar to that described by Kincade et al (13). Mononuclear cells were plated at a density of 5 x 104/ml by mixing with prewarmed Bacto-Agar (Difco Laboratories, Detroit, Mich.) and additional medium or medium supplemented with lipopolysaccharide (LPS) (Difco).…”