Inhibition of agar colony formation by partially purified granulocyte extracts (chalone)

Granulocytic extracts (GE) of different sources, presumably containing the granulocytic chalone, were prepared in different laboratories and purified to some extent. They specifically inhibited the formation of granulocyte and macrophage colonies in agar. The effect was however most pronounced on granulocyte and mixed granulocyte-macrophage colonies, and less on macrophage types. Addition of GE to bone marrow cells at the time of plating in agar, as well as short incubation of the cells together with GE prior to plating, inhibited subsequent colony formation. The inhibitory effect could easily be reversed by washing the cells with an excess of medium prior to plating during the first hour of preincubation, but not after five hours. Increasing the doses of colony stimulating activity (CSA) (at low doses of GE) released the inhibitory effect, but not at high doses of GE. The inhibitory effect of GE on colony formation was dose dependent down to almost 100% inhibition. No apparent cytotoxic effect of GE on bone marrow cells could be found and lymphoblastic cells were not inhibited. Extracts containing a specific inhibitor of erythropoiesis (EIF) stimulated myelopoietic colony formation in agar.     

T-lymphocyte colony formation of murine thymocytes in agar contained in glass capillaries

Optimal growth conditions are presented for a new colony test with mouse thymocytes in agar contained in glass capillaries. The kinetics of colony growth and the dependence from the PHA-, I1-2-, agar- and 2-mercaptoethanol concentration are shown. The colony forming cells are identified as T-lymphocytes by usual morphology and by an indirect immunoperoxidase method using mouse anti-Thy 1.2 antibodies.     

Colony growth of mouse bone marrow cells in agar contained in glass capillaries.

Mouse bone marrow cells were grown in semi-solid agar contained in glass capillary tubes. Several parameters affecting colony formation in the capillaries were studied. 10(4) cells in 100 mul incubation medium within one capillary produced 22 to 30 colonies of granulocytes and macrophages. Compared with the common petri dishes glass capillaries offer several advantages under the conditions used: 1. A twofold higher plating efficiency. 2. Applicability to optical scanning by light scattering and electronic counting, allowing automation and greatly improving sensitivity, statistical accuracy and reproducibility. Kinetics of colony growth can also be monitored. 3. Diminished risk of bacterial and fungal contamination. 4. A more than tenfold lower need for materials on similar statistical errors. Substituting methylcellulose for agar resulted in colonies of fibroblast-like cells adherent to glass surface. Glass non-adherent cells showed a threefold higher plating efficiency in agar.     

Trypanosoma cruzi: colony formation and clonal growth in agar

Trypanosoma cruzi exhibited colonial growth when incorporated into 0.5–0.6% agar. Colonies were established from a single organism and clones readily derived. The plating efficiencies were variable depending on the original inoculum but were consistently over 50% when 10⁴ to 10⁵ parasites were added. The use of this technique for evaluation of an antitrypanosomal agent, nifurtimox, was demonstrated, making possible large-scale testing of potential antitrypanosomal agents and assessment of microbicidal and microbio-static drug levels.     

Autoclaved agar contains an inhibitor of granulocyte-macrophage colony growth in vitro

An inhibitor of granulocyte-macrophage colony growth is produced when agar is autoclaved. We have no evidence that this inhibitor is specific for the granulocyte-macrophage precursor; autoclaved agar may well also be deleterious to the culture of other cell types. We therefore suggest that workers should not use autoclaved agar routinely without first testing it for similar inhibitory actions in their own particular culture systems.     

Improvement of human melanoma colony formation in soft agar using boiled instead of autoclaved agar

The effect of agar sterilized by either boiling or autoclaving on human melanoma colony formation in soft agar was compared using cells from 17 biopsies of metastatic malignant melanoma. The frequency of colony formation was significantly increased for cells grown in boiled agar in 8 samples (47%), unchanged in 8 samples (47%), and decreased in only one sample (6%). There were increases in both cluster and colony formation for the melanomas which had augmented colony formation when grown in boiled agar. There was also qualitative morphological improvement, including rounder, smoother cells and less extracellular debris surrounding the colonies. These data suggest that melanoma colony formation is enhanced when cells are grown in agar which has been sterilized by boiling rather than autoclaving.     

B and T lymphocyte colony formation in agar: 2-mercaptoethanol-activated albumin can substitute for 2-mercaptoethanol.

The effect of 2-mercaptoethanol (2-ME) activated albumin (MaSF) on mouse B lymphocyte colony (BLC) formation and on human phytohemagglutinin (PHA)-induced T lymphocyte colony formation (TLC) formation in semisolid agar medium was studied. MaSF was found to stimulate colony formation comparable to the stimulation obtained with 2-ME. MaSF could not substitute for serum in any of the agar culture systems. BLC formation stimulated by MaSF was obtained with spleen cells from athymic nude mice and nonadherent spleen and lymph node cells of normal mice suggesting a direct effect of MaSF on the colony-forming B cell without interference with T cells or macrophages. The results suggest that the stimulatory effect of 2-ME in the BLC and TLC agar systems is mediated by 2-ME-activated albumin present in the culture medium.     

Fibroblast growth factor stimulates colony formation of differentiated chondrocytes in soft agar

The effect of fibroblast growth factor (FGF) on the growth of chondrocytes in soft agar was examined. FGF induced colony formation by chick embryo and rabbit chondrocytes. The colony-forming efficiency of FGF-exposed chondrocytes was similar to that of Rous sarcoma virus-transformed chondrocytes (15-20%). Other mitogenic agents tested, such as epidermal growth factor, insulin, insulin-like growth factor-l, and platelet-derived growth factor, induced very low levels of colony formation. The induction of growth in soft agar of chondrocytes by FGF was not due to cells' phenotypic transformation, because chondrocytes grown in soft agar with FGF retained the ability to synthesize cartilage-characteristic proteoglycan. FGF did not induce growth in soft agar of chondrocytes whose phenotypic expression was suppressed by retinoic acid or 5-bromodeoxyuridine. In addition, FGF did not induce growth in soft agar of primary fibroblasts and normal rat kidney (NRK) cells. These results suggest that FGF selectively stimulates growth of differentiated chondrocytes in soft agar.     

Stimulation of human T cell colony growth by a lymphocyte colony enhancement factor derived from lymphocyte subpopulations

Blood mononuclear cells (MNC) develop into T cell colonies when the cells are sensitized with PHA and seeded in a two-layer soft agar system. Conditioned medium (CM) derived from MNC enhanced lymphocyte colony formation when it was added to the culture system. CFU-TL appear to be stimulated into colony formation by molecules secreted by lymphocyte subpopulations contained in the seeded cells. In this study, human peripheral blood MNC were fractionated by a battery of techniques into adherent, E+, CD4+, CD8+, B and null cells. CM was prepared from each of the subpopulations and its effects on T cell colony growth assayed. All the lymphocyte subpopulations were found to generate lymphocyte colony enhancement factor (LCEF). After several purification procedures, CM prepared from CD4 and CD8+, displayed LCEF activity corresponding to proteins of molecular weight 30-40 and 100-140 kD.     

Chalone inhibition of granulocyte colony growth in agar: kinetic quantitation by capillary tube scanning.

Mouse bone marrow cells were seeded into capillary tubes containing agar with colony stimulating factor. The development of myelomonocytic clusters and colonies was followed by daily tube scanning using their light scattering properties. Three kinetic scanning parameters were determined and the significance of different threshold settings was evaluated; viz. the number of signals, the mean signal height and the signal integrals. The inhibitory effect of two extracts with known granulocyte chalone activity which had been prepared from human peripheral leukocytes and rat bone marrow cells, was followed with the scanning method. A continuous reduction of clusters and colony formation and their growth throughout the incubation period was observed which suggested a sustained retardation of proliferation of both the stem cells committed for myelomonopoiesis and their progeny.     

Clonal variation in colony morphology and growth of CHO cells cultured on agar

Single Chinese hamster ovary (CHO) cells plated on agar form macroscopic colonies with high efficiency. Colonies produced by cells from the uncloned cell line increase in diameter continuously for 10–12 days after plating to form mounds of cells about 1 mm in diameter. With further incubation, some of these colonies do not increase in diameter (arrested dome), some form an expanding annular monolayer of cells around the central mound (fried egg), and some grow by enlarging the central mound into a low multilayered disc (saucer).These colony types on agar appear to be clonal characteristics of the CHO cell line. Cloning the line gives two kinds of isolates: one forms a mixture of arrested dome and fried egg colonies in an inheritable ratio, and the other forms saucer colonies. Cells from saucer colonies form saucer colonies when replated on agar. Cells from all colony types replate with similar efficiency on plastic or agar, and exhibit the same growth rate and cell size in liquid suspension culture. On plastic substrate, all these CHO cells form colonies which increase continuously in diameter for as long as 21 days, and little clonal difference in the morphology of colonies or of single cells is observed.These observations reveal a previously unsuspected heterogenieity in an established line of cultured mammalian cells and provide a method for studying new classes of In vitro growth control phenomena. These control phenomena may help in the building an in vitro model for tumor growth.     

Regulation of colony formation of differentiated chondrocytes in soft agar by transforming growth factor-beta

Fibroblast growth factor (FGF) induces colony formation by chondrocytes in soft agar (Y. Kato et al., J. Cell. Physiol., 1987), and the present study revealed that transforming growth factor-beta(TGF-beta) does not induce the same effect. TGF-beta did, however, increase the efficiency of colony formation by chondrocytes 3- to 4-fold in the presence of a maximal dose of FGF. Furthermore, TGF-beta decreased the concentrations of FGF needed for the induction of cell growth in soft agar by 40- to 100-fold. These results suggest that TGF-beta is involved in the control of cartilage growth possibly by increasing the responsiveness of chondrocytes to FGF.     

A simple agar plate assay for screening siderophore producer yeasts.

Yeasts produce hydroxamate-type siderophores (iron-binding compounds) in response to Fe-stress conditions. Because these siderophores are important to the biocontrol of postharvest diseases of apple and pears, a method for screening siderophore producer yeast was developed.The screening method was carried out in special Petri dishes with eight or nine wells (25-mm diameter). These wells were filled with siderophore production medium and seeded with yeasts isolated from epiphytic apple microflora. After yeasts grew (24-48 h), holes (2-mm diameter) were made in the agar of each well. Holes were filled with an acid solution of ferric perchlorate. After 10-15 min, reddish halos appeared in the bottom of the plate and their intensities were compared with standards. Standards were prepared in the same special dish with rhodotorulic acid solutions (concentrations between 0.05 and 1 g/l) plus 2% agar. When agar solidified into wells, holes were made and filled with ferric perchlorate solution. Color intensities of reddish halos were proportional to siderophore concentration and the detection limit was 0.1 g/l. It was possible to correlate the production of siderophore in solid medium with the results obtained in liquid medium. The methodology was also a useful tool for making a preliminary assessment of the influence of different factors on the siderophore production.     

One-week 96-well soft agar growth assay for cancer target validation

Soft agar growth, used to measure cell anchorage-independent proliferation potential, is one of the most important and most commonly used assays to detect cell transformation. However, the traditional soft agar assay is time-consuming, labor-intensive, and plagued with inconsistencies due to individual subjectivity. It does not, therefore, meet the increasing demands of today's oncology drug target screening or validation processes. This report describes an alternative 96-well soft agar growth assay that can function as a replacement for the traditional method and overcomes the aforementioned limitations. It offers the following advantages: a shortened assay duration (1 week instead of 4 weeks) that makes transient transfection or treatment possible; plate reader quantification of soft agar growth (measuring cloning efficiency and colony size); and a significant reduction in required labor. Higher throughput also makes it possible to process large numbers of samples and treatments simultaneously and in a much more efficient manner, while saving precious workspace and overall cost.     

Ehrlich ascites cell (EAC) chalone assay using purified calf thymus-DNA polymerase alpha

A relatively rapid chalone assay using inhibition of purified calf thymus DNA polymerase alpha by Ehrlich Ascites Cell (EAC) chalone has been performed. The DNA polymerase alpha was inhibited in a concentration-related fashion by partially purified EAC chalone ranging from 10 to 200 micrograms/ml. Spermidine was also tested since there has been some suggestion that chalone may be spermidine; we found no effect of spermidine at 170 and 230 microM, but marked inhibition at 33 mM. This assay should facilitate chalone purification, since chalone appears to non-specifically inhibit DNA polymerase alpha.