Du 600 spectrophotometer

Ultraviolet–visible light (UV–Vis) spectroscopic measurements were performed using a Beckman DU600 spectrophotometer. Samples were diluted to appropriate concentrations in double-distilled H₂O and placed in a 1 cm cuvette. Fe²⁺—ferrozine complex formation being measured using a 400–800 nm wavelength scan and H₂O₂ was monitored using the absorbance at 240 nm. Fe²⁺—ferrozine and H₂O₂ concentrations were calculated using Beer’s Law using the absorbance at 562 nm (ε₅₆₂ = 27,900 mol⁻¹ cm⁻¹) and 240 nm (ε₂₄₀ = 46.3 mol⁻¹ cm⁻¹), respectively.

ELISA method was used to examine the levels of plasma anti-heart antibody (AHA) including antibodies (Abs) against the adenine nucleotide translocator (ANT), β1 adrenergic receptor (β1-AR), myosin heavy chain (MHC) and L-type calcium channel (CC) according to the procedures described in previous studies13 (link)14 (link)15 (link)16 (link)17 (link). Briefly, the peptides derived from different human cardiac proteins including ANT (PIERVKLLLQ-YDEIKKFV), β1-AR (HWWRAESDEARRCYNDPKCCDFVTN RC), MHC (EIERKLAEKD-VDKLQLKV-AKSRDIGAKGLNE) and CC (VNENTRMYIPEENHQ) were synthesized by a PSSM-8 automated peptide synthesizer (Shimadzu, Japan). The purities of the synthetic peptides confirmed by high liquid chromatography were up to 96%. A horseradish peroxidase-labeled rat antihuman IgG (Gibco, USA) was used to detect AHA, and the absorbance (A) of the dye is measured at a wavelength of 450 nm by using a Beckman DU-600 Spectrophotometer. All of the samples were measured in triplicate.

P. infestans strains were cultured by shaking at 20 °C and then washed twice and diluted to approximately (3 × 10⁴ zoospores/mL with cold 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, pH 6.0. Cells were aliquoted to tubes, and 7a–d, 8a–d, and 9a–d was added at a final concentration of 100 µg/mL. SDS (2%) was used as the reference compound, which produced 100% cellular oomycete leakage. P. infestans was incubated at 20 °C, and samples were taken at time intervals (6, 12, 24, and 48 h) and spun at 3500 rpm for 7 min in microcentrifugetubes. The supernatants were collected for absorbance analysis at 260 nm in a Beckman DU-600 spectrophotometer [37 (link)]. Results are the means of values from at least two independent assays.

Mass production of the plasmid PJW4303, PJW4303-SjC23 and PJW4303-SjC23-mHSP70 DNA vaccines was conducted according to manufacturer’s instructions for QIAGEN-2500. The concentration and purity was detected by the BECKMAN DU-600 Spectrophotometer. The A₂₆₀/A₂₈₀ value of the prepared DNA ranged between 1.85 and 1.95. DNA was resuspended in sterile physiological saline (0.9% NaCl, PH 7.4). All DNAs were diluted to 1 mg/ml.

All urine samples were analyzed by urinalysis using a Siemens CLINITEK® status automated analyzer (Tarrytown, NY) to ensure no contamination or infection. All samples were centrifuged at 4000rpm/680g for 10 minutes to remove cellular debris, aliquoted, and frozen at -80C. Protein quantification was performed, as previously described [17 (link), 18 (link)], using 100ul of thawed urine by Bradford assay (Biorad, Hercules, CA) read on a Beckman DU600 Spectrophotometer.
Human MMP2, MMP7, TIMP1, TIMP2, and NGAL Quantikine ELISAs were obtained from R&D Systems (Minneapolis, MN). Aliquots of 1ml were thawed and run in duplicate for each ELISA protein target according to the manufacturer’s protocol for urine. Optical density was measured at 450nm with a correction at 540nm using a FilterMax F3 Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA). Dilution series optimization was performed for each ELISA to ensure that sample readings would be within the upper and lower limits of detectability based on the standard curve. The following dilutions were utilized: MMP-2 and TIMP-1 undiluted, MMP-7 1:5, NGAL 1:10, and TIMP-2 1:2. Protein quantifications in ELISA were measured in concentration (ng/ml) and then normalized to total protein (pg/ug) as standard practice.

Duplicate water samples (400 mL each) were filtered onto 25 mm diameter 0.4 μm pore size polycarbonate filters and stained for 2 s with a 0.02% aqueous solution of Alcian blue in 0.06% acetic acid. The filters were stored at -20°C until analysis. For analysis the filters were soaked in 5 mL of 80% sulfuric acid and the absorbance was measured at 787 nm in 1 cm disposable polystyrene cuvettes using a DU 600 spectrophotometer (Beckman Coulter), and using ultrapure water as blanks. Three blanks were performed in each batch of samples every day (including staining and freezing in parallel to the samples). Each solution of Alcian blue was calibrated using a fresh standard solution of gum xanthan (GX) with a concentration of 25 mg L^-1. The coefficient of variation of the replicates was ~17%. TEP concentrations (μg of gum xanthan equivalents per liter -μg GX Equiv L^-1-) were measured according to Passow and Alldredge [30 (link)].