Clonal Proliferation of Human Stimulated Lymphocytes on Agar Culture

Rozenszajn, L. A.; Shoham, D.; Kalechman, I.

doi:10.1007/978-1-4613-4355-4_19

Cited by 38 publications

(48 citation statements)

References 15 publications

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“…The present communication establishes that some peripheral blood leucocytes are capable of clonal expansion in agar into colonies with T lymphocyte characteristics and confirms the original observation of Rozenszajn et al (1974).…”

Section: Resultsmentioning

confidence: 47%

Human T Lymphocyte Colonies in Agar: A Comparison With Other T Cell Assays in Healthy Subjects and Cancer Patients

Wilson

Dalton

1976

Aust J Exp Biol Med

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Summary Colonies of human lymphocytes with T cell characteristics will grow in agar from repeated mitotic divisions with phytohaemagglutinin (PHA) stimulation. The colonies comprise spheres of tightly‐packed cells with up to 500–1,000 blast‐like cells in each colony. 65% of cells from pooled colonies bound AET‐treated sheep red cells. 1,100–2,500 colonies/106 peripheral blood lymphocytes developed when cell donors were healthy but lower numbers (350–1,000 colonies/106 lymphocytes) were detected in blood from cancer patients. Comparison with other non‐specific assays of cell‐mediated immunity showed that, while 66% of cancer patients were anergic (to five recall antigens) and 78% exhibited depressed mitotic activity in standard cultures with low dose PHA, 100% of these patients revealed T cell colony formation below normal. It is suggested that further studies of T lymphocyte colony‐forming cells in healthy people and in a number of disease states may significantly advance our understanding of mechanisms of cell‐mediated immunity.

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Section: Resultsmentioning

confidence: 47%

Human T Lymphocyte Colonies in Agar: A Comparison With Other T Cell Assays in Healthy Subjects and Cancer Patients

Wilson

Dalton

1976

Aust J Exp Biol Med

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“…Both phenomena have previously been described for liquid culture systems (6)(7)(8). Resistance to alkali treatment, absolute dependence on the presence of mercaptoethanol, differential sensitivity of the stimulated cells to anti-/~ antibodies, and different patterns of Ig class expression by proliferating cells further distinguishes agar mitogen from LPS.1 Agar has been used by others as a supporting matrix for studying cell-cell interactions in mitogen responses (9), to clone mitogen-activated cells (10)(11)(12), and in culture systems where agar was assumed to be inert (13). Our findings show that mitogenic stimulation by constituents of common agar can be avoided by either using agarose as a supporting material or by omitting 2-ME from the culture medium.…”

Section: Discussionmentioning

confidence: 44%

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

Kincade¹,

Ralph²,

Moore³

1976

The Journal of Experimental Medicine

104

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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 *

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“…In the mouse, B lymphoid colonies are formed in the presence of 2-mercaptoethanol (5), and T lymphoid colonies can be induced with the plant lectins phytohemagglutinin (PHA) and concanavalin A (6). T lymphoid colonies can also be established from human peripheral blood lymphocytes in the presence of PHA (7)(8)(9), whereas with pokeweed mitogen mixed T and B lymphoid colonies are formed (9). Established human lymphoid cell lines multiply spontaneously in the absence of plant lectins or mercaptoethanol, and it seemed possible that such cells might release growth-stimulating substances into the culture medium.…”

mentioning

confidence: 43%

Lymphocyte culture: induction of colonies by conditioned medium from human lymphoid cell lines.

Galbraith¹,

Goust²,

Fudenberg³

1977

The Journal of Experimental Medicine

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It has been shown that macrophage and granulocyte colonies can be induced m semisolid agar (1-3) in the presence of substances termed colony-stimulating factors (CSF), which are released predominantly by monocytes (4). However, attempts to induce formation of lymphoid colonies with CSF have so far proved unsuccessful. In the mouse, B lymphoid colonies are formed in the presence of 2-mercaptoethanol (5), and T lymphoid colonies can be induced with the plant lectins phytohemagglutinin (PHA) and concanavalin A (6). T lymphoid colonies can also be established from human peripheral blood lymphocytes in the presence of PHA (7-9), whereas with pokeweed mitogen mixed T and B lymphoid colonies are formed (9). Established human lymphoid cell lines multiply spontaneously in the absence of plant lectins or mercaptoethanol, and it seemed possible that such cells might release growth-stimulating substances into the culture medium. We have therefore investigated the ability of conditioned medium (CM) obtained from lymphoid cell lines to induce normal human peripheral blood lymphocytes (PBL) to form lymphoid colonies in agar. Materials and MethodsThree B-cell lines-4061 (provided by Dr. J. Sinkovics, Houston, Tex.), PGLC 33H, and RPMI 1788-were used as sources of CM. After 3-4 days of culture in serum-free medium at 37°C, cellfree supernates were obtained by centrifugation at 800 g for 10 rain, filtration through 0.22-~m filters (Millipore Corp., Bedford, Mass.), and ultracentrifugation at 47,500 g for 1 h.Peripheral blood was collected from adult healthy volunteers in pyrogen-and preservativefree heparin. Lymphocytes were obtained by centrifugation over Ficoll Isopaque gradients and washed three times in RPMI 1640 medium (Grand Island Biological Co., Grand Island, N.Y.). Contamination with monocytes varied between 3 and 20% as assessed by latex ingestion and staining for peroxidase (10). Cells were seeded at various concentrations in 2 ml 0.3% agar made by the addition of I vol of double-strength RPMI 1640 (containing 200 gg/ml streptomycin, 200 U/ ml benzyl penicillin, and 20% fetal calf serum [Gibco]) to 1 vol of 0.6% agar (Difco Laboratories, Detroit, Mich.) made up in pyrogen-free distilled water. Various amounts of CM were added directly to the 0.3% agar mixture. After culture in 35-ram plastic Petri dishes at 37°C in 5% CO2 in air for various times, the plates were counted under an inverted microscope. Colonies were defined as clumps containing more than 50 cells. Colony cells were tested for their ability to form E rosettes, by using the technique of Wybran et al. (11), and for the presence of surface * Publication no. 145 from the

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Clonal Proliferation of Human Stimulated Lymphocytes on Agar Culture

Cited by 38 publications

References 15 publications

Human T Lymphocyte Colonies in Agar: A Comparison With Other T Cell Assays in Healthy Subjects and Cancer Patients

Human T Lymphocyte Colonies in Agar: A Comparison With Other T Cell Assays in Healthy Subjects and Cancer Patients

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

Lymphocyte culture: induction of colonies by conditioned medium from human lymphoid cell lines.

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