Fluorescence (excitation 485 nm; emission 535 nm) was measured with the use of a microplate reader. RNA was isolated from Qiazol suspended cells according to the manufacturer’s protocol and quantified spectrophotometrically. Reverse transcription Etoposide datasheet reaction was performed using 500 ng of RNA, which was reverse-transcribed into cDNA using iScript™ cDNA synthesis kit (Biorad, Veenendaal, The Netherlands). Next, real time PCR was performed with a BioRad MyiQ iCycler Single Color RT-PCR detection system using Sensimix™Plus SYBR and Fluorescein (Quantace-Bioline, Alphen a/d Rijn, The

Netherlands), 5 μl diluted (10 × ) cDNA, and 0.3 μM primers in a total volume of 25 μl. PCR was conducted as follows: denaturation at 95 °C for 10 minutes, followed by 40 cycles of 95 °C for 15 seconds and 60 °C for 45 seconds. After PCR, a melt curve (60–95 °C) was produced for product identification and purity. β-actin was included Selleck MDV3100 as internal control. Primer sequences are shown in Table 1. Data were analyzed using the MyIQ software system (BioRad) and were expressed as relative gene expression (fold change) using the 2ΔΔCt method. 1.1E7 cells were incubated with 0.5 mM CML for 24 hours. After incubation,

culture medium was collected. Cytokines released in the supernatant of the cells were measured using the Bio-plex pro assay according to manufacturer’s instructions. This assay uses antibodies coupled to magnetic beads which react with 50 μl supernatant. After a series of washes to remove unbound protein, acytokine-specific biotinylated detection antibody was added to the reaction. After 30 minutes incubation and several washes, a streptavidin-phycoerythrin (streptavidin-PE) reporter complex was added to bind biotinylated detection antibodies. The plate was then read using the Luminex system and data was analyzed using the Bio-Plex Manager software™.

1.1E7 cells were incubated nearly with 0.5 mM CML for 24 hours. After incubation, cells were washed with PBS, harvested with trypsin-EDTA and centrifuged (1000xg, 5 minutes, 4 °C). Next, cells were washed with ice-cold PBS and centrifuged again. Cell pellets were then resuspended in ice-cold extraction buffer (0.1% Triton X-100 and 1.3% SSA in a 0.1 M potassium phosphate buffer with 5 mM EDTA, pH 7.5) and sonicated in icy water for 10 minutes. The extracts were used for determination of intracellular GSH and GSSG content using an enzymatic recycle method described by Rahman et al. [18]. 1.1E7 cells were incubated with 0.5 mM CML for 24 hours. After incubation, cells were washed with HBSS, harvested with trypsin-EDTA and centrifuged (1000xg, 5 minutes, 4 °C). Cell pellets were then resuspended in 145 mM sodium phosphate buffer pH 7.4 containing 1 mM EDTA. Next, cells were sonicated in icy water for 10 minutes and centrifuged (15 minutes, 10.000xg, 4 °C). Final reaction mixture (1 ml) contained 0.06 mM NADPH (in 1% Na2CO3) and 50 μl sample in buffer. The reaction was started by the addition of 0.225 mM GSSG (in 0.