Fetal bovine serum (FBS), phytohaemagglutinin, and trypsin–EDTA were purchased from Cutilab (Campinas, SP, Brazil). RPMI 1640 medium selleck chemicals llc was purchased from GIBCO (Invitrogen, Carlsbad, CA, USA). ConA, Rhodamine 123

(Rho-123), etoposide, penicillin, streptomycin, and MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). Normal melting point agarose (NMPA) and low melting point agarose (LMPA) were obtained from Invitrogen (Carlsbad, CA, USA). Doxorubicin (Doxolem®) was purchased from Zodiac Produtos Farmacêuticos S. A. (São Paulo, SP, Brazil). All other chemicals and reagents used were of analytical grade. ConA was obtained from SIGMA (São Paulo, Brazil) and ConBr was purified from the crude saline extract of seed flour through affinity chromatography on Sephadex G-50 fast flow (SIGMA) according to Cavada et al. (1998). The human promyelocytic leukemia

(HL-60) and acute lymphoblastic cell (MOLT-4) lines selleck compound were acquired from Rio de Janeiro Cell Bank (Federal University of Rio de Janeiro, RJ, Brazil). Leukemia cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C with 5% CO2. For experiments, the concentration of FBS was reduced to 1% so that the lectins would display their effects (Faheina-Martins et al., 2011). Heparinized blood (from healthy, non-smoking NADPH-cytochrome-c2 reductase donors who had not taken any drugs for at least 15 days prior to sampling) was collected from donor blood at the blood bank of the João Pessoa, Paraíba, Brazil. From these blood samples, we isolated the peripheral blood mononuclear cells (PBMC). The study was approved by the Institutional Ethical Committee

of Lauro Wanderley Hospital/Federal University of Paraíba. PBMC were isolated by a standard method of density-gradient centrifugation over Histopaque-1077 (GE Healthcare, USA). PBMC were washed and resuspended in RPMI 1640 medium supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C with 5% CO2. Phytohemagglutinin (2%) was added at the beginning of the culture. After 24 h of culture, cells were treated with the test lectins. The cytotoxicity of ConA and ConBr to leukemic cells was evaluated using the original enzymatic reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to produce formazan crystals (Mosmann, 1983). Cells were seeded at 5 × 104 cells/well in 96-well tissue culture plates. Cells were exposed to different concentrations of ConA or ConBr lectins (1–200 μg/mL) dissolved in the RPMI medium (three wells per concentration) with 1% FBS. After 72 h of incubation, plates were centrifuged (500g, 5 min) and the supernatant was removed, followed by the addition of MTT solution (0.5 mg/mL in PBS) and incubation for 4 h at 37 °C. After 4 h, the MTT formazan product was dissolved in SDS/HCl 0.