Du 64 spectrophotometer

Chromatography of Human Serum on DEAE Cellulose, pH 7: Ion exchange chromatography of human serum was performed on a 12-mL DEAE cellulose column (Whatman Specialty Products, Fairfield, NJ, United States) equilibrated in 50 mM NaCl, 10 mM phosphate, pH 7.5. Three milliliter of human serum dialyzed in equilibration buffer was added to the column, washed with seven column volumes of buffer, and collected in four equal fractions (zero absorbance in fourth fraction). A linear 50-mL salt gradient of 50–1,000 mM NaCl (in 10 mM phosphate, pH 7.5) was used to elute proteins from the column, and 0.6-mL fractions were collected. The salt concentration was monitored with an osmometer. The protein concentration was measured at 280 nm with a Beckman DU/64 spectrophotometer with a micro cuvette. Between 85 and 90 percent of the IL-8 secretory activity of primary non-CF nasal epithelial cells was found in the pre-gradient wash buffer fractions; these fractions were pooled and concentrated.

ROS-induced lipid peroxidation was examined via measuring the level of 4-hydroxy-2-nonenal (4HNE, an end-product by lipid peroxidation) in accordance with previously described methods [29 (link),30 (link)]. In brief, five gerbils per group were deeply anesthetized by intraperitoneal injection of 90 mg/kg pentobarbital sodium (JW Pharm. Co., Ltd., Seoul, Korea). Thereafter, their brains were harvested and hippocampal CA1 tissues were dissected. The obtained CA1 tissues were homogenized with a buffer containing 10 mM Hepes (pH 7.5, containing 200 mM mannitol, 70 mM sucrose, 1 mM EGTA, and 5 mM butylated hydroxytoluene) and extracted with dichloromethane. An aliquot of the lower organic phase was dried under nitrogen and rehydrated. The samples were mixed with N-methyl-2-phenylindole in acetonitrile and methane sulfonic acid. Thereafter, they were incubated and centrifuged. Using a Beckman DU-64 spectrophotometer (Beckman Instruments, Inc., Fullerton, CA, USA), the absorbance of the clear supernatant was measured at 586 nm wavelength. The 4HNE concentration in each sample was determined against 4HNE standard provided in the assay kit.

The molecular size of the active protein from the previous steps was determined with a 12-mL S-200 Sephacryl gel column (GE17-0584-10, Sigma). The column was run with PBS. Two hundred microliters of concentrated protein was layered onto the gel, and 150-μL fractions were collected. The protein concentration was measured at 280 nm with a Beckman DU/64 spectrophotometer with a micro cuvette.

A stock of P. gingivalis (ATCC 33277) was received from the Department of Oral Implantology, Tokyo Medical and Dental University. The bacteria were propagated and streaked using a sterile loop onto a Vital Media Brucella HK Agar RS (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan) and anaerobically incubated using the AnaeroPack System (Mitsubishi Gas Chemical, Tokyo, Japan) at 36 °C with 4.6% of CO₂ for 96 h. A few colony units of P. gingivalis from the plate were inoculated into a test tube of anaerobic bacterial culture medium (ABCM) (Eiken Chemical Co., Ltd., Tokyo, Japan), resulting in a density of 2.0 × 10⁷ colony forming units per millimeter (CFU/mL), and anaerobically incubated for another 96 h. After the incubation period, a standard curve correlating optical density and bacterial concentration was created based on a serial dilution of the incubated bacteria. The absorbance was measured at a wavelength of 600 nm (OD₆₀₀) using a DU-64 spectrophotometer (Beckman Coulter, Brea, CA, USA).

The absorbance values of the samples and the blank were determined at 535 nm using a (Beckman DU-64 spectrophotometer, USA) and then blank absorbance value was subtracted from the sample absorbance value. From a standard curve, MDA concentration in the unknown sample was extrapolated from the corresponding absorbance using the regression line from the standard curve and expressed as nmol/gm tissue by multiplying in the tissue dilution factor.

A few colony units of P. gingivalis from the culture agar were inoculated into a glass test tube of ABCM (OD₆₀₀ = 0.15) and anaerobically incubated with the AnaeroPack System. Bacterial solution (100 μL) was inoculated together with SeNPs at various concentrations (900 μL) in glass test tubes and incubated for 96 h. Blank solutions for the control group were prepared with ABCM without bacteria and without SeNPs. Blank solutions for the experimental groups were prepared by adding SeNPs of each concentration into ABCM without bacteria. After the indicated incubation period, 130 μL of solution was removed from well-vortexed bacteria/SeNP solution and placed into a cuvette for OD₆₀₀ measurements using a Du-64 spectrophotometer (Beckman Coulter, Brea, CA, USA). The measured optical density value was extrapolated to bacterial concentration (CFU/mL) using the standard curve initially created with the Du-64 spectrophotometer.