The evaluated parameters included cell membrane integrity, internucleosomal DNA fragmentation, cell cycle, mitochondrial depolarization, phosphatidylserine (PS) externalization and caspase 3/7 activation. For all the

tested compounds, five thousand events were evaluated per experiment, and cellular debris was omitted from the analysis. HL-60 cell GSK2126458 solubility dmso fluorescence was then determined by flow cytometry in a Guava EasyCyte Mine® using Guava Express Plus software. Internucleosomal DNA fragmentation and the cell cycle were analyzed by ModFit LT for Win32 version 3.1. The experiments were performed in triplicate. To verify the participation of ROS in the quinone activity, NAC (5 mM) was pre-incubated with the cells for 1 h prior to drug addition, and after 24 h, cell membrane integrity, internucleosomal DNA fragmentation and phosphatidylserine (PS) externalization were measured, as previously described. During the apoptotic

process, DNA is cleaved in a distinctive way at internucleosomal sites by a specific caspase-activated endonuclease, thus yielding fragments in multiples of 200 bp, which appear as a characteristic “ladder” when DNA is separated by gel electrophoresis (Enari et al., 1998). Fragmented DNA was isolated as described by Ausubel et al. (1990), using DNAzol® Reagent (Gibco® – Invitrogen, Carlsbad, CA, USA) after 24 h of incubation. buy GSK2118436 Electrophoresis was performed in a 1.5% agarose gel. The alkaline comet assay was performed as described by Singh et al. (1988) with minor modifications. Briefly, HL-60 cells were incubated for 3 or 24 h with five concentrations of QPhNO2 (0.5, 1.0, 2.0, 5.0 or 10 μM) and with nor-beta at 2.0 or 10 μM. Then, the cells were processed and dissolved in 0.75% low melting point agarose and immediately spread onto a glass microscope slide pre-coated with a layer of 1% normal melting point agarose. The slides were further incubated in ice-cold lysis solution (pH 10.0) at 4 °C for at least 1 h.

After the lysis procedure, the slides were placed in a horizontal electrophoresis unit filled with enough fresh buffer (300 mM NaOH and 1 mM EDTA, pH ∼13.0) to cover the slides for 20 min at 4 °C. Electrophoresis was conducted for 20 min at 25 V (300 mA). Idelalisib cell line The slides were then neutralized (0.4 M Tris, pH 7.5) and fixed with ethanol 100%. After the staining step with ethidium bromide, the gels were dried at room temperature overnight, and 50 cells from each of two replicate slides were selected and analyzed for each concentration of test substance. These cells were scored visually into five classes according to tail length: (1) class 0: undamaged, without a tail; (2) class 1: with a tail shorter than the diameter of the head (nucleus); (3) class 2: with a tail length 1–2× the diameter of the head; (4) class 3: with a tail longer than 2× the diameter of the head; and (5) class 4: comets with no heads.