PHA-induced proliferation of human blood mononuclear cells The isolated PBMC were distributed into 96-well flat-bottom plates in 100 µL aliquots (2 × 105 cells/well). PHA was added at a concentration of 5 µg/ml. The compounds were tested at doses of 1, 10, and 50 µg/ml. DMSO at selleck appropriate dilutions served as control. After a four-day incubation in a cell culture incubator, the proliferative response of the cells was determined by the colorimetric MTT method (Hansen et al., 1989). The results are given in percentage inhibition as compared with appropriate
DMSO controls. Cytotoxicity of the compounds against Ilomastat cell line human blood mononuclear cells PBMC at density of 2 × 105/well, re-suspended in the culture medium, were cultured for 24 h in a cell culture incubator with the preparations at indicated concentrations. Cell survival was determined by MTT colorimetric method (Hansen et al., 1989). The results are given in percentage inhibition as compared with appropriate DMSO controls. Lipopolysaccharide-induced TNF-a production in whole blood cell culture Venous blood from a single donor was diluted 10× with RPMI-1640 medium and distributed in 1 ml aliquots in 24-well culture plates. The cultures were stimulated by addition of 1 µg/ml of LPS from E. coli, O111:B4. The compounds were added to the cultures at concentrations of 5 and 25 µg/ml. Higher
concentrations of the compounds could not be used because of inhibitory effects on TNF-α production by corresponding DMSO (the solvent) dilutions. Appropriate dilutions of DMSO served as controls. After overnight incubation this website in a cell culture incubator, the supernatants were harvested and frozen at −20 °C until cytokine determination by a biological assay (Espevik and Nissen-Meyer, 1986). The results are given in percentage Lck inhibition as compared with appropriate DMSO controls. Growth inhibition
of tumor cell lines L-1210 lymphoma and SW-948 colon tumor cell lines derived from the Collection of Cell Lines of The Institute of Immunology and Experimental Therapy, Wrocław, Poland. The lines were re-suspended in the culture medium and distributed into 96-well flat-bottom plates. L-1210 was present at 1.5 × 104 cells/well while SW-948 and at 2.5 × 104 cells/well. The preparations were added to the wells at the concentration range of 0.1–50 µg/ml. Cisplatin was used as a reference drug in the same concentrations. After 3-day incubation in a cell culture incubator, the proliferation was determined using MTT colorimetric method. The data are presented as a mean OD value from quadruplicate wells ± SE. Statistics The results are presented as mean values ± standard error (SE) or percentage inhibition = [(control value − tested value)/control value] × 100. Brown-Forsyth’s test was used to determine the homogeneity of variance between groups.
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