agar and Lymphoma

Topics: Agar; Aneuploidy; Animals; Chromosome Aberrations; Cloning, Molecular; Leukemia L5178; Lymphoma; Mice; Mutagenicity Tests; Mutagens; Polymorphism, Restriction Fragment Length; Time Factors

Current concepts of immunotherapeutic approaches in leukemias and lymphomas using activated cytotoxic lymphocytes and macrophages are briefly reviewed. Defective cellular immunocytotoxic activities and effects of interleukins and chemotherapeutic drugs thereupon are discussed. In vitro assays to measure lymphokine-activated killer (LAK) and natural killer (NK) cell activities suffer from various problems, depending on the quality of the endpoints. Our clonogenic microassay for LAK cell activity, using agar-containing glass capillaries, avoids some of the potential artifacts and offers several advantages that are discussed. As an example the stimulatory effect of low mafosfamide concentrations on the LAK cell activity versus K562 human myeloid leukemia cells is demonstrated. Thus, our clonogenic LAK microassay provides a valid tool for preclinical screening of immunomodulatory agents.

Topics: Agar; Cyclophosphamide; Cytotoxicity Tests, Immunologic; Cytotoxicity, Immunologic; Humans; Immunotherapy, Adoptive; Interferons; Interleukins; Killer Cells, Lymphokine-Activated; Killer Cells, Natural; Leukemia; Lymphoma; Tumor Cells, Cultured; Tumor Stem Cell Assay

Topics: Agar; Animals; Child; Culture Media; Culture Techniques; Hodgkin Disease; Humans; Inclusion Bodies, Viral; Leukemia; Lymphoma; Lymphoma, Large B-Cell, Diffuse; Lymphoma, Non-Hodgkin; Male; Mice; Microscopy, Electron; Mycoplasma; Neoplasms; Neoplasms, Experimental; Rabbits; Rats

Mycoplasmal infection of cell cultures remains a significant threat to diagnostic and research procedures. In certain defined situations, curing of mycoplasmal infected cultures is a reasonable exercise. Four methods of curing were compared: treatment with BM-cycline, 5 bromouracil, use of specific antisera and treatment of infected cells suspended in soft agar with antibiotics. Antisera treatments were of low efficiency of curing: 50%. None of nine infected cell lines treated with 5-bromouracil were consistently cured of mycoplasmas. The use of BM-cycline was effective for some, but not all lines and required long periods of treatment, 12-21 days. 35 naturally or deliberately infected cultures were treated in soft agar a total of 119 times. This procedure which consisted of suspending infected cultures in soft agar containing appropriate antibiotics resulted in successful mycoplasmal elimination 118/119 times. This soft agar technique took 1-3 days. In separate studies, it was shown that certain Mycoplasma fermentans strains were resisted to this and other curing methods. This may be due to their intracellular location. Such strains may be more amenable to antibiotics that penetrate mammalian cells. It is concluded that the soft agar technique is a rapid, efficient and reliable method to eliminate cell culture mycoplasmas.

Topics: Agar; Animals; Bromouracil; Cells, Cultured; Fibroblasts; Humans; Lymphocytes; Lymphoma; Melanoma; Methods; Mice; Monocytes; Mycoplasma; Tumor Cells, Cultured

The L5178Y TK+/- mouse lymphoma assay is widely used in mutagenicity testing. Trifluorothymidine-resistant (TFTr) mutants are quantitated following growth in agar-supplemented cloning medium. In an attempt to evaluate the effect of agar on plating efficiency, we have tested several lots of Difco Noble agar (cat. No. 0142-01-8; normally used in this assay) and compared it with Baltimore Biological Laboratory (BBL) agar (cat No. 11849). We find that BBL agar gives a higher and less variable plating efficiency than any of the Noble lots tested. Colonies plated in BBL agar tend to appear significantly earlier on the plates than those cloned in Noble agar. The absolute mutant number and the induced mutant frequency quantitated from a treated culture is generally higher in BBL compared to Noble agar. To determine if this higher frequency is due to increased mutant recovery rather than "sneak through" of nonmutant cells, we isolated 97 mutants from treated cultures (44 large colonies and 53 small colonies) and 69 mutants from untreated cultures (24 large colonies and 45 small colonies) and tested them for TFT resistance. All but one (a large colony from an untreated culture) were found to be TFTr, indicating that the mutant frequency is due to an increased mutant recovery. The spontaneous mutant frequency was quantitated for 122 untreated cultures. Showing little variation within and between experiments, the spontaneous mutant frequency yielded a mean of 57.7, with a standard deviation of 14.4. Under our laboratory conditions, BBL agar gave reliable results, and we prefer it for use in cloning L5178Y mouse lymphoma cells.

Topics: Agar; Animals; Cell Division; Cell Survival; Lymphoma; Methyl Methanesulfonate; Mice; Mutagenicity Tests; Quinolines; Thymidine Kinase

TFTr mutants of L5178Y/TK+/- mouse lymphoma cells are analyzed as they appear in situ following cloning and incubation for 9-11 days in soft agar cloning medium. These TFTr mutants can be divided by colony size into sigma, small colony, and lambda, large colony, mutants. The use of a size discriminator on an automatic colony counter allows the production of histograms to evaluate the size distribution of colonies on a plate. The evaluation of these size distribution curves provides insight into the properties of sigma and lambda mutants. From these analyses several conclusions may be drawn. The sigma phenotype is preferentially associated with the TFTr subpopulation of a treated culture. The sigma phenotype is not an artifact of delayed toxicity following treatment. The frequency of quantifiable sigma mutants is not affected by agar concentrations between 0.20% and 0.45% in the cloning medium. TFTr sigma mutants are produced spontaneously and can be induced by a variety of mutagens. The decline in overall detectable mutants frequency observed for some mutagens with increasing time after treatment is due to the decline in sigma mutant frequency. The quantitation of both sigma and lambda mutants is thus useful in obtaining maximum utility of the information provided by the L5178Y/TK+/- mouse lymphoma assay.

Topics: Agar; Animals; Bromodeoxyuridine; Cell Division; Cell Survival; Culture Media; Dose-Response Relationship, Drug; Drug Resistance; Kinetics; Lymphoma; Mice; Mutation; Phenotype; Thymidine; Thymidine Kinase; Trifluridine

This study demonstrated the importance of the methods used in determining the lymphoma cell colony stimulating activity of factors derived from lymphoma cells. The in vitro colony formation in a semisolid matrix of the AKR mouse lymphoma cell line, SL 12, and three cloned derivatives, SL 12.1, SL 12.3, and SL 12.4, was studied. We show that the use of soft agar or methylcellulose as a semisolid matrix results in colony formation by the lymphoma cells only in the presence of serum. The addition of conditioned medium (CM) from lymphoma cells growing in serum-free medium does not stimulate colony growth. However, when purified agarose is used, colonies grow in a dose-dependent manner in the absence of serum and in the presence of CM. These results indicate that the type of semisolid matrix used can influence results in studies of this nature. Purified agarose provides the best environment when colony formation by lymphoma cells is used to measure the presence of growth factors in test-conditioned media.

Topics: Agar; Animals; Cell Division; Cell Line; Clone Cells; Colony-Forming Units Assay; Culture Techniques; Lymphoma; Methylcellulose; Mice

The effect of 11 anticancer drugs on the ability of Raji lymphoma cells to form colonies in soft agar was determined with the use of both a 1-hour and a continuous drug exposure. Three distinct patterns of drug sensitivities were observed: a) Dactinomycin, adriamycin, bleomycin, mitomycin C, vincristine, and cis-platinum II all produced a dose-dependent reduction in colony formation following a 1-hour exposure, which was further augmented by a continuous exposure to the drugs; b) the antimetabolites (methotrexate, beta-cytosine arabinoside, and 5-fluorouracil) and pentamethylmelamine had no suppressive effects on colony formation with a 1-hour exposure, but they produced marked cytotoxicity with continuous drug exposure; and c) L-phenylalanine mustard had the same degree of colony suppression with both a 1-hour and a continuous drug exposure. Preincubation of Raji cells with an enzyme mixture (DNase + pronase + collagenase) did not alter the degree of colony suppression observed with the anticancer drugs. These results indicate that continuous drug exposure should be compared to a 1-hour drug incubation to determine in vitro drug sensitivities of fresh human tumors in the soft agar clonogenic assay, because the 1-hour drug exposure may not identify certain drugs that are potentially clinically active.

Topics: Agar; Antineoplastic Agents; Cell Line; Cell Survival; Clone Cells; Colony-Forming Units Assay; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Humans; Lymphoma; Time Factors

Growth of cells from patients with lymphoma was promoted by feeder layers containing medium conditioned by adherent spleen cells of mineral-oil-primed BALB/c mice or by cells from a human B lymphocyte line (RPMI 1788). Sixty-five patients with all histologic types of non-Hodgkin lymphoma were studied. Lymphoid colony growth was obtained in 61% of bone marrows and 50% of lymph nodes histologically involved by lymphocytic lymphoma. Conversely, colony growth was observed in only a single instance from 49 bone marrows without overt lymphoma and was not observed in cultures of normal lymph nodes, spleens, bone marrows, peripheral blood, or thymuses. Colonies appeared within four days of plating and reached peak size in 7--10 days. Plating efficiency ranged from 0.001% to 0.1%. Morphological, histochemical, and immunological studies of cells from the colonies identified them as lymphoid, and sufficient evidence is available to designate the colony-forming units as putative lymphoma stem cells.

Topics: Agar; Bone Marrow; Cell Adhesion; Cell Division; Cells, Cultured; Clone Cells; Culture Media; Humans; Lymph Nodes; Lymphoma; Spleen

Topics: Acridines; Agar; Analysis of Variance; Animals; Cell Count; Cell Line; Cells, Cultured; Clone Cells; Dexamethasone; Drug Resistance; Gamma Rays; Genetic Variation; Lymphoma; Mice; Mice, Inbred BALB C; Mutagens; Nitrosoguanidines; Radiation Effects

Topics: Agar; Animals; Autoradiography; Cell Line; Culture Media; Culture Techniques; Leucine; Lymphoma; Mice; Microscopy, Electron; Microscopy, Electron, Scanning; Microscopy, Phase-Contrast; Time Factors; Tritium; Uridine

Topics: Agar; Carcinoma; Electrophoresis; Gels; Half-Life; Hodgkin Disease; Humans; Isoenzymes; L-Lactate Dehydrogenase; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Leukocytes; Lymphatic Diseases; Lymphoma; Neoplasms; Osteosclerosis; Plasmacytoma; Sarcoma; Tissue Extracts

Topics: Agar; Chemical Precipitation; Humans; Hydrolysis; Hypergammaglobulinemia; Immunoelectrophoresis; Immunoglobulin G; Lymphoma; Multiple Myeloma; Pectins; Polysaccharides

The technique involved in studying the activation of lymphocytes in the resting form, and their recognition as dividing and functional cells was studied, using phase contrast and agar as well as fluid culture. Standardization of technical methods was found to be essential, and the effect of variables was studied. Lymphocytes from human umbilical cord vein blood were found to be spontaneously activated. Infestation by microfilaria either diminished or entirely inhibited activation. The significance of lymphocytic cohesion was considered and the formation of colonies of activated lymphocytes on agar is described. The effects of non-human vertebrate lymphocytes and of human cells other than lymphocytes were studied. Spontaneous activation of abnormal lymphocytes was noted.

Topics: Agar; Animals; Chickens; Cricetinae; Culture Techniques; Filariasis; Haplorhini; Humans; Lymphocytes; Lymphoma; Methods; Microscopy, Phase-Contrast; Mitosis; Rabbits; Rats; Umbilical Veins