After the incubation, the cells were centrifuged at 10,000g for 3

After the incubation, the cells were centrifuged at 10,000g for 30 min at 4 °C, and the supernatant was collected and filtered through a 0.2 μm Millipore membrane. The absorbance was determined in a spectrophotometer DU-800 (Beckman Nutlin3a Coulter, Fullerton, CA, USA) by the difference in absorbance at wavelengths 414 and 600 nm. The results are expressed in nmol cytochrome c released/106 cells using a molar extinction coefficient (ɛ) of 100 mM−1 cm−1. The assessment of caspase 3 activity was performed using a Caspase 3 assay kit (Sigma–Aldrich). The hepatocytes

were collected and centrifuged at 600g for 5 min and suspended in 1 mL of phosphate buffered saline (PBS). Further centrifugation was performed, and the precipitate was incubated for 15 min at 4 °C

with 200 μL of lysis buffer for the release of caspase 3, and 300 μL of PBS was then STA-9090 added. The lysed cell suspension was centrifuged at 14,000g for 15 min at 4 °C, and the supernatant was collected. Aliquots of 50 μL of supernatant were used to assess the activity of caspase 3 according to the manufacturer’s instructions. Fluorescence was determined using the fluorescence spectrophotometer RF-5301 PC (Shimadzu, Tokyo, Japan) at wavelengths of 360 and 460 nm for excitation and emission, respectively. The results are expressed as pmol of AMC/min/mL. Samples of cells (200 μL) were collected and centrifuged at 50g for 5 min, and the precipitate was suspended in Krebs/Henseleit medium, pH 7.4, and incubated with Hoechst 33342 (8 μg/mL) and Propidium Iodide (5 μM) dyes for 15 min at room temperature in the dark. After incubation, the samples were

centrifuged very twice at 50g for 5 min to remove excess dye. After the washes, the hepatocytes were suspended in 50 μL of Krebs/Henseleit medium, pH 7.4. The cells were analyzed with a fluorescence microscope (DM 2500 type, Leica, Rueil-Malmaison, France), and the percentage of necrotic cells was quantitated using the Qwin 3.0 software. Data are expressed as the mean ± standard error of the mean (S.E.M.). The statistical significance of the differences between control and the experimental groups was evaluated using one-way analysis of variance (ANOVA) followed by Dunnett’s test, and differences between the experimental groups at the same time points was evaluated using unpaired t test with Welch´s correction. Values of P < 0.05 were considered to be significant. All statistical analyses were performed using GraphPad Prism software, version 4.0 for Windows (GraphPad Software, San Diego, CA, USA). Fig. 1 shows the inhibitory effect of ABA on the glutamate-plus-malate-supported and succinate-supported state 3 (ADP-stimulated) respiration of mitochondria in digitonin-permeabilized hepatocytes.

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