Clusters In Suspension Research Articles

A new method for measuring clustering in suspension between accessory cells and T lympocytes

Specifying the molecular basis and clinical significance of cluster formation between antigen-presenting cells and T lymphocytes will be important in many areas of immunology. In this paper we describe a novel and reproducible technique for measuring cluster formation in suspension between purified human blood monocytes and purified autologous T lymphocytes, and its application to determining the effects of recall antigens and mitogen. Blood monocytes and T lymphocytes from eight normal subjects were separately prelabelled with two different carbocyanine dyes prior to co-culture in suspension with or without antigen (PPD, SKSD) or mitogen (PHA). At 24 h the co-cultures were examined for cluster formation by ultraviolet microscopy and flow cytometry. Control experiments showed that the carbocyanine dyes were non-toxic in vitro, that cell labelling was stable for culture periods up to 120 h, and that the two dyes did not leak from cell to cell. By this technique we measured the proportion of monocytes clustering one or more T lymphocytes in the presence and absence of recall antigen or PHA. There was a close correlation between visual and flow cytometric measurement of monocyte: T lymphocyte clustering ( p < 0.001) as well as a close relationship between the ability of the two recall antigens to increase the extent of clustering above the baseline ( p < 0.001). Antigen-increased cluster formation did not correlate with baseline clustering, unlike PHA-increased clustering, which was related to baseline levels ( p = 0.02), suggesting the operation of distinct mechanisms. The method is applicable to measuring cell-cell associations in suspension during extended periods of culture, as well as for the study of agents which might modify intercellular adhesion processes.

Induction of B16 melanoma melanogenesis by a serum-free synthetic medium

Cultured marine B16 melanoma cells normally grow as spindle-shaped cells firmly attached to tissue culture flasks. Pellets obtained from harvested B16 melanoma cells are white to grey in color. When the same cells were grown in synthetic, serum-free AIM V medium, cellular morphology and pigmentation were radically altered. Within 3 days of subculture in AIM V, cells rounded up and grew in clusters in suspension. Melanin content increased to greater than 30 times and tyrosinase activity was found to be 10–50 times higher in cells grown in AIM V medium compared to those cultured in normal medium. A concomitant increase in the level of immunoreactive tyrosinase was also induced. The individual growth factors and hormones present in AIM V medium were examined to determine which component(s) stimulates melanogenesis. Only those cells grown in the presence of 2.5% human albumin were stimulated to synthesize melanin. These findings suggest that albumin, or a component associated with albumin, has a major effect upon the regulation of melanogenesis in these cells.

Establishment and characterization of cell lines from human small cell and large cell carcinomas of the lung.

Five new small cell carcinomas (SCC) cell lines and a large cell carcinoma (LCC) cell line were established from human lung cancers. The SCC cell lines had, as a group, common phenotypic properties which distinguished them from non-SCC cell lines. However, the studies also revealed a considerable biological heterogeneity among the individual SCC cell lines. Thus, the SCC cell lines had a typical growth pattern with cell clusters in suspension or partly adherent to the bottom. All the lines examined grew in agarose with variable cloning efficiencies, and all but one line formed tumors subcutaneously in nude mice. The ultrastructure of the SCC cell lines was characteristic with dense core granules at a variable frequency. Neuron-specific enolase was detectable in all SCC cell lines, usually in large amounts, and an inconstant production of a spectrum of polypeptide hormones was found, typical of SCC. The LCC cells proliferated in monolayers, formed colonies in agarose and grew in nude mice. Ultrastructurally, the LCC cell line differed from the SCC cell lines in having intra- and intercellular lumina and tonofilaments. The capacity of the LCC and a previously established squamous cell carcinoma cell line (U-1752) to produce neuron-specific enolase and polypeptide hormones was characteristically much lower than that of the SCC cell lines. We conclude from this study that SCC cell lines, although individually distinct from one another, are quite homogeneous as a group in expressing a set of basic common neuro-endocrine markers. However, these studies also suggest some biological relationship between SCC, LCC and SQC by virtue of their expression of some common neuro-endocrine markers, in support of the concept of a common histogenetic origin of human lung cancers.

Serum-free growth of normal and transformed fibroblasts in milk: differential requirements for fibronectin.

Bovine milk may be used as a supplement for the serum-free growth of certain fibroblastic cells in culture. The growth properties of three representative cell types in milk-supplemented medium were examined; fibroblastic cell strains, fibroblastic cell lines, and transformed fibroblasts. Transformed fibroblasts, which included RNA and DNA tumor virus-transformed cells and carcinogen-transformed cells, grew in milk. Instead of growing attached to the culture dishes, as they normally do in serum, transformed fibroblasts grew in milk as large clusters in suspension. In contrast, nontransformed fibroblastic cell strains and cell lines did not grow in milk-supplemented medium. Fibroblasts transformed by a temperature-sensitive transformation mutant of Rous sarcoma virus were temperature-sensitive for growth in milk. The failure of cells to adhere to the substratum in milk-supplemented medium suggested that milk might be deficient in attachment factors for fibroblasts. When the attachment of fibroblastic cells in milk-supplemented medium was facilitated by pretreating culture dishes with fibronectin, (a) transformed cells grew attached rather than in suspension, (b) normal cell lines attached and grew to confluence, and (c) normal cell strains adhered and survived but did not exhibit appreciable cell proliferation.