Transmembrane mitochondrial potential was evaluated by incorporating Rhodamine-123 (Rho-123), which is a cell-permeable, cationic, fluorescent dye that is readily sequestered by active mitochondria without inducing cytotoxic effects. Treated and Sorafenib untreated leukemic cells during 24 h were centrifuged at 500g for 5 min and the pellet was resuspended in 200 μl of a 1 μg/ml solution of Rho-123 for 15 min in the dark. After incubation, cells were centrifuged at 500g for 5 min. The resulting pellet was resuspended in 200 μl of phosphate-buffered saline (PBS) and incubated for 30 min in the dark. Fluorescence was measured and the mitochondrial depolarization percentage was determined.
For all cytometric experiments, cell fluorescence was determined by flow cytometry in a Guava EasyCyte Mini (Guava Technologies, Inc., Hayward, CA, USA) using Guava Express Plus software, as described by Logrado et al. (2010). Five thousand events were evaluated per experiment. The intracellular ROS was
estimated by fluorescent probe, 2´,7´-dichlorohydrofluorescein diacetate (H2-DCF-DA). This dye is deacetylated by intracellular esterase and converted to non fluorescent 2´,7´-dichlorohydrofluorescein (H2-CDF), which is rapidly oxidized to the highly fluorescent compound www.selleckchem.com/products/dabrafenib-gsk2118436.html 2´,7´-dichlorohydrofluorescein (DCF) in the presence de ROS. The MOLT-4 and HL-60 cells (4 × 105 cells/well) were treated with lectins ConA or ConBr (5, 25 and 50 μg/ml). As a positive control H2O2 (50 μM) was used for 15 min. After 24 h of exposure, the samples were centrifuged (200g, 5 min), washed with PBS at 37 °C and labeled of H2-DCFH-DA for 30 min, in the dark conditions, at 37 °C.
The cells were then washed Alanine-glyoxylate transaminase with PBS and analysed by flow cytometry in BD FACSCalibur, NJ, USA ( Ravidran et al., 2011). The data are expressed as mean ± SEM from three replicates per treatment. Results were analyzed by one-way ANOVA followed by Newman-Keuls post-test. The level of significance was set at p < 0.05. Data of all the results in this study were obtained from at least three independent experiments. The correlation between numbers of late apoptosis cells, expressed as arbitrary unit of DNA damage, was performed using the least squares linear regression, f = ax + b, where a means the slope of the line and b determines the point at which the line crosses the y-axis. The Pearson’s correlation coefficient (r), was considered to be significant when p < 0.05. As displayed in Table 1, the MTT-based assay and total nucleic acid content (NAC) measurements show that ConA and ConBr lectins have cytotoxic effects in leukemic cells. The MOLT-4 cell line was more sensitive to exposure of ConA and ConBr than the HL-60 cells were after 72 h of treatment. Among the tested lectins, ConBr was much less active than ConA in the MTT assay.