, 2011). Results are expressed as a percentage of fluorescence intensity with respect to the control. An oxidation system comprising 2,2′-azino-di (3-ethylbenzthiazoline-6-sulfonic

acid) (ABTS), myoglobin and hydrogen peroxide (H2O2) has been used for TAC assay to determine Trolox equivalent antioxidant capacity (Kambayashi et al., 2009 and Yu and Ong, 1999). We used this assay to assess the antioxidant capacity of PFT. Briefly, 90 μL of 10 mM phosphate buffered saline (pH 7.2), 50 μL of myoglobin solution, 20 μL of 3 mM ABTS solution, and 20 μL of PFT or Trolox solution were added to 96-well selleck products microplates. Reactions were started by addition of H2O2 (final concentration: 250 μM), and were followed at 600 nm with a microplate reader for 10 min. Cells were seeded into the Lab-Tek® 8-well chambered cover glass system (Thermo Fisher Scientific, Inc.) at densities of 2 × 104, and were incubated overnight under standard culture conditions. Cells

were pre-treated with or without PFT at 20 μM for 1 h, followed by incubation with DHA at 120 μM for the indicated times. Chambered slides were washed twice with phosphate buffered saline (PBS). For detection of protein 1 light chain 3 (LC3), cells were fixed in ice-cold 1:1 methanol:acetone for 30 min. Slides were immersed for 50 min in 1% goat serum and 0.1% Triton X-100 in PBS, and were then transferred to 10% goat serum/PBS for 20 min. Following the click here PBS rinse, slides were selleck chemicals llc incubated with primary antibody (anti-LC3; MBL, Nagoya, Japan) at 1:1000 in PBS for 1 h at room temperature, washed with PBS, and then incubated with fluorescein isothiocynate (FITC)-conjugated anti-rabbit secondary antibody (Beckman Coulter, Brea, CA) for 30 min. For detection of cytochrome c, after incubation with reagents, the medium was removed and cells were fixed in Mildform® (Wako, Osaka, Japan) for 10 min. Slides were immersed for 5 min in 0.1% Triton X-100 in PBS and were then transferred to 3% FBS/PBS for 30 min. After washing with PBS, slides were incubated with Alexa Fluor® 555 mouse anti-cytochrome c antibody (BD Pharmingen™, San Jose, CA)

at 1:40 in PBS for 1 h. After incubation with antibodies, rinsing with PBS and a drop of UltraCruz™ Mounting Medium with DAPI (Santa Cruz Biotechnology, Inc., Dallas, TX) was added to each well. Cells were observed under a confocal fluorescence microscope (C-1; Nikon, Tokyo, Japan) for blue fluorescence intensity (405 nm) indicating the nucleus, green fluorescence intensity (488 nm) indicating LC3-positive cells (indicative of autophagy), or red fluorescence intensity (562 nm) indicating expression of cytochrome c. In order to detect the effects of PFT and DHA on mitochondrial membrane potential (ΔΨM) in HepG2 cells, we used the Cell Meter JC-10 mitochondrial membrane potential assay kit (AAT Bioquest®, Inc., CA).