Garamycin

The PBMCs were used to verify whether treatment with CPBMF65 would influence viability or induce toxicity in normal human cells. The PBMCs were isolated from the blood of healthy humans by gradient centrifugation on Ficoll-Paque (GE Halthcare, USA). A total of 12 mL of heparinized blood was diluted 1:2 with saline solution. Then, 2 mL of Ficoll-Paque was added to 6 mL of the previous dilution and centrifuged at 720 x g and 22ºC for 20 min. PBMCs were collected (cloudy layer) from the diluted blood/Ficoll-Paque interface. Afterward, the cell pellet was washed twice in 10 mL of phosphate buffered saline (PBS). Cells were re-suspended in Roswell Park Memorial Institute (RPMI 1640; Gibco, Lifetechnologies, USA), supplemented with 0.15% garamycin (Schering-Plough, USA) and 20% homologous serum at a final density of 1.6 × 10 6 cells/mL and maintained in humidified incubator at 37 °C and 5% CO 2 [11] (link). All experiments were performed in triplicate.

Octyl gallate (Sigma-Aldrich), LPS-Escherichia coli 026:B6 (Sigma-Aldrich), Ficoll-Paque TM PLUS (GE healthcare), NH 4 Cl (Nuclear), Heparin sodic (Hepamax-S), RPMI 1640 medium (Gibco-Life Technologies), Quanti-it TM PicoGreen ® dsDNA assay kit (Invitrogen), Trypan blue dye (Gibco-Life Technologies), 2,2-diphenyl-1-picrylhydrazyl-DPPH (Sigma-Aldrich), 96-well microtiter bottomed flat plates (Nunc TM-Immuno Modules), Garamycin (Schering-Plough), Phytohemagglutinin (PHA) (Invitrogen), Vitamin C (Sigma-Aldrich), Phosphate buffer saline-PBS (Laborclin), 2′,7′-Dichlorofluorescin diacetate-DCFH-DA (Sigma-Aldrich), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-MTT (Gibco-Life Technologies).