The cells were washed with PBS and incubated with streptavidin-horseradish CB-839 research buy peroxidase for 10 minutes. After rinsing with PBS, the cells were immersed in DAB solution. The cells were counterstained for 3 minutes with 1% methyl green. Cells containing fragmented nuclear chromatin characteristic of apoptosis will exhibit brown nuclear staining that may be very dark after labeling. Detection of lactate dehydrogenase (LDH) activity The conversion of lactate to pyruvate was detected using the Cytotoxicity Detection Lactate Dehydrogenase

kit (Roche Applied Science, IN, USA) following the manufacturer’s instructions. MCF-7 breast cancer cells and PBMC treated with colloidal silver were washed twice with ice-cold PBS, harvested by GDC-0973 supplier centrifugation at 250 g for 10 min at 25°C, and the supernatant was used for the activity assay according to the manufacturer’s instructions. Optical densities resulting from LDH activity were measured in a microplate reader at 490 nm. Results were given as the mean + SD of three independent experiments. Nitrite determination Accumulation of nitrite in the supernatants of control and treated MCF-7 and PBMC cultures was used as an indicator of nitric oxide production. Cells were

incubated for 5 h in DMEM/F-12 medium, in the presence or absence of colloidal silver in triplicates, in a total volume of 200 μL DMEM/F-12 medium. After incubation, supernatants Idasanutlin were obtained and nitrite levels were determined with the Griess reagent, using NaNO2 as standard. Optical densities at 540

nm were then determined in a microplate reader (Bio-Tek Instruments, Inc.). Determination of intracellular antioxidants The antioxidants production was measured using the following kits: Cellular glutathione peroxidase (Gpx) assay kit (Oxford Biomedical Research, MI, USA), superoxide dismutase (SOD) assay kit (Cayman Chemical Company, MI, USA), and catalase (CAT) assay kit (Cayman Chemical Company, MI, USA) according to the manufacturer’s instructions. Briefly, to determine the activity of Gpx, SOD, and CAT; MCF-7 and PBMC were incubated with LD50 (3.5 ng/mL) and LD100 (14 ng/mL) of colloidal silver for 5 h. Cells Cell press were then washed three times with PBS and sonicated on ice in a bath-type ultrasonicador (80 Watts output power) for 15-s periods for a total of 4 min; the solution was then centrifuged at 1500 g for 5 min at 4°C. The obtained supernatants were used to determine intracellular antioxidants in a microplate reader at 540 nm. Total antioxidant (extracellular antioxidants) The total antioxidant production was determined using the Total Antioxidant Colorimetric Assay Kit (US Biological, Massachussets, USA) following manufacturer’s instructions. Briefly, MCF-7 and PBMC were treated with LD50 (3.5 ng/mL) and LD100 (14 ng/mL) of colloidal silver for 5 h. Thereafter, supernatants were used to determine antioxidants in a microplate reader at 490 nm.