Uv 2550

ROS are extremely short-lived and highly reactive; hence, their exact measurement in tissue samples remains difficult [53 (link),83 (link)]. Here, measurements of MDA (a secondary product) and H₂O₂ (an important ROS) were used as indicators of ROS levels [84 (link)]. MDA and H₂O₂ concentrations in protein supernatants of thoracic aortic samples were measured using MDA and H₂O₂ assay kits (Nanjing Jiancheng, Nanjing, China), respectively, according to the manufacturer’s protocol. The supernatant from the thoracic aorta protein extract was mixed with the analytical reagent containing thiobarbituric acid (TBA), and the mixture was heated at 100 °C and cooled to room temperature. Then, the mixture was centrifuged at 3000 rpm for 15 min at 4 °C. The absorbance of the supernatant was then measured by spectrophotometry (Shimadzu UV-2550, Kyoto, Japan) at 532 nm, and finally, MDA concentration in the thoracic aorta was determined by comparing its OD532 value with the MDA standard value. H₂O₂ reacts with molybdic acid to form stable molybdic acid peroxide compounds, and absorbance was measured spectrophotometrically at 405 nm (Shimadzu, UV-2550, Kyoto, Japan), and finally, the H₂O₂ concentration in the thoracic aorta was determined by comparing its OD405 value with the H₂O₂ standard value.

Endogenous H₂O₂ concentrations were determined according to Patterson et al. [28 (link)]. Hydrogen peroxide coupled with titanium sulfate generating superoxide-titanium which was a yellow precipitate. Superoxide-titanium was dissolved by sulfuric acid, and the color of the solution had a linear relation with hydrogen peroxide concentration. The absorbance was read at 415 nm using an ultraviolet-visible spectrophotometer (UV-2550, Shimadzu, Japan). H₂O₂ was determined from a calibration curve.
Accurately measured 0.5 g of C. roseus leaves for the determination of peroxidase activity (POD, EC 1.11.1.7) was homogenized to a fine powder under liquid nitrogen. Then, the enzyme was extracted from 5 mL PBS (pH 7.0) with 1.0 mM EDTA, 1.0 mM ascorbic acid, and 10 g/L PVP. The homogenate was centrifuged under −4°C and 10000 g/min for 30 min. A hundred-microliter sample solution was added to 1.8 mL hydrochloric acid buffer solution, 1.0 mL guaiacol, and 0.1 mL hydrogen peroxide. Peroxidase activities were assayed in UV-2550 (Shimadzu, Japan) at 460 nm for 3 min [29 (link)].

Apterous adult aphids were homogenized in TRIzol reagent (Invitrogen, USA). The extracted RNA samples were treated with DNase (RNase free) (NEB, USA) to exclude DNA contamination. The total RNA was analyzed with gel electrophoresis and quantified using a spectrophotometer (UV-2550; SHIMADZU, Japan). The first-strand cDNA was synthesized according to the manual of the SuperScript III first-strand synthesis system for RT-PCR kit (Invitrogen, USA). All of the available nucleotide sequences of the A. gossypii CarE gene (Genbank No. AY485218 and Genbank No. AY485216) were retrieved from the NCBI GenBank database, and a homology search to define the conserved regions was carried out using Megalign (DNASTAR) software. The forward primer 5′-taatacgactcactataggg TAACCCTTGGGCGTTTACTG-3′and the reverse primer 5′-taatacgactcactataggg GGTCTCGTCGCAAAAATCAT-3′ were used to amplify a CarE gene fragment. A 686 bp fragment was amplified and confirmed by sequencing. The fragment was used as a template to generate the corresponding dsRNA using the MEG Ascript RNAi kit (Ambion, USA). dsRNA-CarE was dissolved in 50 µL diethypyrocarbonate (DEPC)-treated water, analyzed with gel electrophoresis (1% agarose), and quantified using a spectrophotometer (UV-2550; SHIMADZU, Japan).

We measured the absorbance of different concentrations of LV at 282 nm and 353 nm using an ultraviolet spectrophotometer (Shimadzu UV-2550, Kyoto, Japan) and established a standard curve. Two stock standard solutions (100 µg/mL) were prepared by dissolving 10 mg of LV in 100 mL of PBS (pH 7.4) and hydrochloric acid (pH 1.2). In addition, the stock standard solution was diluted with PBS and hydrochloric acid to yield seven standard solutions containing varying concentrations of LV (5, 10, 15, 20, 25, 30, and 35 µg/mL) to be used in the linearity assessment. We measured the absorbance of the standard solution with an ultraviolet–visible light (UV–Vis) spectrophotometer (Shimadzu UV-2550, Kyoto, Japan) and made a standard curve. The in vitro release was studied using a dialysis bag method. Briefly, LV@MSN–COOH and LV@MSN–COOH/GC were placed in dialysis membrane bags (molecular weight cut-off (MWCO) 8–14 kDa, Solarbio, Beijing, China) and immersed in a fresh dissolution medium of PBS (pH 7.4) or HCl (pH 1.2). The entire system was placed in a shaker incubator (Shanghai Jing Hong Laboratory Instrument Co., Ltd., Shanghai, China) set at 100 rpm at 37 °C. At predetermined time intervals, 5-mL samples were withdrawn and replaced with equal volumes of fresh media to maintain the sink condition. The sample was quantitatively analyzed at 282 nm or 353 nm over a period of 2 h.

Fresh V. faba seedling leaves (0.5 g) were homogenized in 5 mL of extraction solution containing 50 mM ice-cold phosphate buffer (pH 7.8), 1 mM EDTA and 1% PVP. The sample was centrifuged at 13,200 rpm for 20 min at 4°C, and subsequently, the supernatant was used to assay protein content as well as SOD and CAT activities.
Protein content was measured according to the method of Bradford50 (link) and standardized to bovine serum albumin. The absorbance was measured at 595 nm.
SOD activity was determined according to the method of Song et al.23 (link). The reaction system (3 mL) contained 50 mM phosphate buffer (pH 7.8), 750 μM NBT, 130 mM methionine, 20 μM riboflavin, 0.1 mM EDTA, and 50 μL of the enzyme extract. After illumination at 5000 lx for 15 min, the absorbance of the mixture was measured at 560 nm using an ultraviolet/visible spectrophotometer (Shimadzu, UV-2550).
CAT activity was measured according to the method of Song et al.48 (link). The reaction system (3 mL) contained 100 mM potassium phosphate buffer (pH 7.0), 20 mM H₂O₂ and 0.1 mL of the enzyme extract. The decrease in absorbance at 240 nm was recorded for 1 min using an ultraviolet/visible spectrophotometer (Shimadzu, UV-2550).

The oxidation of linoleic acid by SI was assayed by ultraviolet absorption spectroscopy^{61 (link),62 (link)}. Briefly, 10 μl of linoleic acid at ≥99% purity (Sigma-Aldrich, Missouri, USA) was mixed with 490 μl of NaIO₃ in PBS at indicated concentrations followed by 30 min of incubation at 37 °C. All tubes were filled with nitrogen to prevent oxygen in the air from oxidizing linoleic acid. After the reaction, 900 μl of purified chloroform (Knowles, Chengdu, China) was added to extract the linoleic acid. The absorbance of linoleic acid was recorded with UV–visible spectrophotometer (UV2550, Shimazu, Japan) in the wavelength range of 260 nm-330 nm.

In a typical experiment, different DESs (10%, v/v) co-solvent mixtures (20 ml) or aqueous Tris-HCl buffer (50 mM, pH 5.0) containing Acetobacter pasteurianus GIM1.158 cells (25 mg mL⁻¹) were incubated in a Erlenmeyer flask at 30 °C and 180 rpm. The cell-free supernatants (2.0 ml) containing DESs and released intracellular components were withdrawn from the co-solvent system at 0 h (controls with DESs) and 24 h. The OD₂₆₀ and OD₂₈₀ values of the samples were determined using ultraviolet spectrophotometer (Shimazu UV-2550). The OD₂₆₀ and OD₂₈₀ values at 24 h were corrected for the absorbance of the DESs by subtracting the corresponding 0 h absorbance values, hence these corrected values for the 24 h samples were a measure of the release of intracellular components (primarily nucleic acids and proteins) into the medium during incubation with DESs.
For the flow cytometry (FCM) assay, the cell was pretreated with various DESs according to our previous report32 . After that, the cell suspension was diluted to be 10⁶ cfumL⁻¹ and dyed with propidium iodide (a membrane impermeable fluorescent nucleic acid stain) at 4 °C in the dark, and then was subject to a BD FACS Verse Coulter to determine the cell membrane integrity. The fluorescent emission was excited at 535 nm, and recorded at 550–600 nm. Data from FCM were analyzed using BD FACSuite software.