Nylon wool column: a tool for obtaining monokine-rich eluates in the absence of serum

Diverse conditions for stimulating human mononuclear cells to release thymocyte costimulatory factors were tested for their contribution to the generation of supernatants high titers of these monokines. Activity titers increased with LPS concentration, reaching a plateau between 1 and 10 microng/ml. Indomethacin did not modify the monokine, but the assay for thymocyte costimulatory activity was substantially affected by inhibitory substances produced by the monocytes in the absence of indomethacin. The use of nylon wool columns to trap the cells was shown to be effective in raising cellular densities without decreasing activity titers. As result, the yield per cell could be maintained even in the absence of serum, an important step toward the goal of purifiying bioactive from crude broths

Jacalin: an excellent lectin for obtaining T cell growth activity from rat spleen cells

The parameters involved in the choice of an optimal T cell growth activity (TCGAc) induction protocol using rat spleen cells simultated with jacalin were studied. In the absence of serum, 5 microng/ml jacalin was sufficient to obtain maximal TCGAc, Supernatants could be harvested at any time between 24 and 72 h since significant consumption of TCGAc was not observed during this interval. TCGAc recovery was increased in the presence of 5% fetal calf serum, with the optimal jacalin dose being about 25 microng/ml. The recommended harvesting time was 24 h to reduce TCGAc loss due to cellular proliferation. Human or rat sera were not suitable since they absorb significant amounts of jacalin, thus shifting the optimal lectin concentration to > 800 microng/ml. Indomethacin (1 microng/ml) had little enchancing effect on TCGAc production by rat cells but rendered conditioned media less inhibitory of cytotoxic T lymphocyte L (CTLL) proliferation. Addition of 50 ng/ml phorbol myristate acetate is not recommended if the supernatants are to be used for T cell line maintenance, since the agent interferes with CTL function, while only doubling TCGAc production. Jacalin-stimulated TCGAc recovery is comparable, in titer, to that obtained with concanavalin A under the best conditions, but the former is less expensive due to the large quantities of lectin recovered from a single jackfruit, besides being less toxic for rat spleen cells

Sugar inhibition of the lectin jacalin: comparison of three assays

1. Three assays were used to test nine sugars for inhibition of jacalin activity prepared from Artocarpus intefrifolia. Rat spleen proliferation was unssuitable since the measurement of the effects of sugars against jacalin binding was complicated by their simultaneous metavolic effects on the cells. 2. Based partly on a sheep red blood cell hemagglutination assay and mainly on human serum protein preciptation, the following potencies in relation to D(+)-galactose (taken as 1) were obtained: 1-0-methyl-alfa-D-galactopyranoside, 40; methyl-alfa-D-mannopyranoside and D(+)-mannose, 0.12; ß-Dd-(-)-fructose, 0.08; alfa - D(+)-glucose and 1 - 0-methyl-ß-D-glucopyranoside, <0.04