Thermo varioskan flash spectrophotometer

GSNO reductase activity was measured spectrophotometrically at 340 nm by the time-dependent oxidation of NADH and reduction of GSNO, as described by Sakamoto et al. [22 (link)]. Yeast cells were harvested from the cultures grown in liquid YPD medium to OD₆₀₀ 0.5–1.0 and washed three times with sterile distilled water. The cells were resuspended in 150 μl extract buffer [50 mM Tris-HCl, pH 8.0, and 0.1% (v/v) Tween 20], and mechanically broken with acid-washed glass beads in Fastprep-24 Instrument (MP Biomedicals, Solon, OH, USA). Then samples were centrifuged at 4°C for 10 min at 12,000 x g to remove the beads and insoluble material. The concentration of protein extracts was determined by a Bradford assay, with bovine serum albumin (BSA) as the standard. About 10 μg proteins were incubated in 100 μl assay buffer that contains 20 mM Tris-HCl (pH 8.0), 0.2 mM NADH and 0.5 mM EDTA. The reaction was started through adding GSNO (Cayman Chemical, Ann Arbor, MI, USA) at a final concentration of 400 μM. The absorbance was measured at 340 nm at room temperature using a Thermo Varioskan Flash spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The resultant GSNO reductase activity was expressed as nmol NADH consumed min⁻¹ (mg protein)⁻¹.

Gills tissue approximately 43 ± 4.5 mg was sampled from each bivalve. Malondialdehyde was measured based on the method previously reported for oyster (C. gigas) and mussel (M. edulis) tissues [40 (link), 43 ]. Tissues were homogenized in 5 volumes of buffer (20 mM Tris, 150 mM NaCl, 10 mM β-mercaptoethanol, 20 μM leupeptin, 2 μM aprotinin and 100 μM benzamidine) and centrifuged at 30,000 g for 30 min (4 °C). The supernatant was denatured for 15 min at 75 °C. Since thiobarbituric acid reactives (TBARs) has been criticized for its reactivity towards other compounds, two blanks were incorporated to reduce potential background interference; 1) the gill sample plus the buffer alone without TBARS and 3) the TBARS plus chlorothalonil without gill sample. Subsequently, the absorbance of TBARs in the supernatant was measured at 535 nm in a Thermo Varioskan Flash spectrophotometer (Thermo Fisher Scientific, Tewksbury, MA, USA) using a malonaldehyde bis (1,1,3,3-Tetramethoxypropane, Sigma-Aldrich, Inc.) standard. The blank values were subtracted from entire readings. The concentrations of the lipid peroxidation compounds were expressed as nM of MDA per g of gill tissue.

After dissection, gill tissues from three oysters were pooled as one replicate. Three replicates (totally nine oysters dissected for each group; seawater control, DMSO treatment and exposed groups) was independently analyzed. Approximately 62 ± 2.05 mg (n = 20) of gill tissues was used for each oyster. We followed a previously reported MDA analysis assay for C. gigas gill tissues [25 ]. Tissues were homogenized in 5 volumes of buffer (20 mM Tris, 150 mM NaCl, 10 mM β-mercaptoethanol, 20 μM leupeptin, 2 μM aprotinin and 100 μM benzamidine). After centrifugation for 30 min at 30,000 g (4°C), supernatants were heat-denatured for 15 min at 75°C. Thiobarbituric acid reactives (TBARs) were measured at 535 nm with a Thermo Varioskan Flash spectrophotometer (Thermo Fisher Scientific, Tewksbury, MA, USA) using malonaldehyde bis (tetrametoxypropan, Sigma-Aldrich, Inc.) as standard. The concentration of lipid peroxidation compounds was expressed as nM of MDA per gram of gill tissue.