Spectramax m3 spectrophotometer

The modulation of pro-inflammatory cytokine TNF-α secretion induced by IAPs was evaluated by ELISA. Murine peritoneal macrophages from C57BL/6 mice were pre-treated or not with LPS (500 ng/ml) for 4h and then stimulated or not with different concentrations of Hs IAPs (0.1, 1 and 10 μM) and incubated for 24 h. As a positive control, cells were stimulated only with LPS (500 ng/ml) for 24h. Supernatant TNF-α concentration was detected by ELISA with a R&D Systems (USA) kit. Microtiter plates were coated overnight at room temperature with capture antibody and blocked with Reagent Diluent for 1 hour. Serially diluted samples were added to the wells in triplicate and incubated overnight at 4˚C. After extensive washing, the cells were incubated with detection antibody and then Streptavidin-HRP. After washing, substrate solution was added and the plates were incubated for 15 minutes at room temperature. Plates were read after adding the stop solution at 450nm using SpectraMax M3spectrophotometer (Molecular Devices).

Cell viability was assessed using the crystal violet staining assay,^[28] at room temperature, as an end‐point measure of total biomass generated over the course of the culture period. Briefly, out of 200 µL of medium per well, 150 µL was discarded. To each well, 150 µL of 99.9% methanol (MilliporeSigma) was added for 15 min to kill and fix the cells, then discarded. Afterward, 100 µL of 0.5% crystal violet (25% methanol) was added for 20 min, then the wells were emptied. Each well was washed twice with 200 µL of phosphate‐buffered saline for 2 min. Absorbance (optical density) was acquired at 570 nm using the SpectraMax M3 Spectrophotometer (Molecular Devices) and SoftMax Pro software (Version 7.0.2, Molecular Devices).

Thiobarbituric acid reactive substances (TBARS) are commonly used as a marker of lipid peroxidation, an indirect assessment of oxidative stress. Levels of TBARS were examined in diaphragm muscle homogenates from sham and CIH-exposed mice (n = 8 per group). A standard curve was curated using malondialdehyde (MDA). Fifty μL of thiobarbituric acid (TBA, 50  mM) was added to 50 μL of diaphragm muscle homogenate. The solution was then incubated at 97 °C for 1 h on a dry heating block. Samples were immediately cooled on ice, and 75 μL of methanol: 1  mM NaOH (91:9) was added to the mixture. Samples centrifuged at 704× g. 70 μL of the resultant supernatant was added in duplicate per well in a black 96-well plate. A spectraMax-M3 spectrophotometer (Molecular Devices, USA) using 523/553 excitation/emission settings as used to read the 96-well plate. Data are expressed as nM TBARS per mg of protein.

A 59-peptide array covering the amino acid sequence of the N protein as 17 or 13 mer with 10-amino-acid overlaps was obtained from BEI Resources (catalog no. NR-52404). One milligram of lyophilized peptide was resuspended in 1 mL phosphate-buffered saline (PBS) and diluted to 5 μg/mL in PBS, and 100 μL was added to each well of a 96-well microtiter plate (Nunc-Immuno MicroWell 96-well solid plates; Fisher Scientific). Plates were stored at 4°C for 24 to 48 h and washed three times with 250 μL PBS plus 0.05% Tween 20 (PBS-T). Plates were blocked with 200 μL PBS-T plus 2% bovine serum albumin for 4 h. Blocking buffer was removed and replaced with 100 μL patient sera diluted 1:1,000 in blocking buffer. Plates were incubated for 1 h at room temperature and then washed three times with PBS-T. One hundred microliters of 1 μg/mL horseradish peroxidase (HRP)-conjugated anti-human IgG in blocking buffer was added to wells and incubated for 1 h at room temperature with shaking. The plate was washed three times with 250 μL PBS-T. We added 100 μL 3,3′,5,5′-tetramethylbenzidene (TMB) substrate to wells for 1 min, and then reactions were stopped with 100 μL stop solution. Absorbance was read at 450 nm on a Molecular Devices SpectraMax M3 spectrophotometer using SoftMax Pro v6.5.1 software.

The ability of all the 127 bacterial isolates to form biofilms was evaluated by the method described by Stepanović et al. [53 (link)]. The method essentially utilized crystal violet to identify those bacteria, which attached firmly onto the polystyrene surface of a 96-well flat-bottomed microplate. Briefly, the overnight-maintained bacterial culture was diluted with TBS, which contained 1% glucose with a factor of 1:100. All the diluted suspensions (200 μ L) were incubated in a 96-well microplate for 24 h at 37 °C without shaking. After that, the contents of each well were washed 3 times with water under vigorous shaking. The adherent bacteria were fixed with 200 μ L of 99% methanol for 15 min. Subsequently, the microplates were emptied and were left to dry. The adherent bacteria in each well were then stained with 0.2 mL of crystal violet (2%) for 5 min followed by rinsing with water prior to air-drying. After that, 160 μ L of 33% glacial acetic acid was added into each well to solubilize the adherent bacteria. The OD₅₇₀ of each well was determined by a Spectramax M3 spectrophotometer (Molecular Devices, USA). S. aureus ATCC 43300 was used as a positive control, whereas negative control wells contained the TBS medium only. The cut-off OD (OD_C) was expressed as 3 standard deviations above the mean OD₅₇₀ of the negative control. The biofilm producers were categorized as below:

A stock solution containing 0.8 mg/ml ThT (Acros) in 10× PBS was filtered through a 0.22 µm syringe filter (Millex). Peptide samples were prepared in a black 96-well plate (Corning) to reach a final concentration of 500 µM total peptide, 0.08 mg/ml ThT, and 1× PBS. Samples were analyzed using a SpectraMax M3 spectrophotometer (Molecular Devices) using excitation 450 nm and emission 482 nm. All samples were run in triplicate, with the mean and standard deviation reported.

At sacrifice, a 10 cm portion of small intestine was excised distally from 10 cm below the ligament of Treitz and flushed with ice-cold phosphate-buffered saline (PBS). The intestinal segment was ligated at one end and a tube was inserted to add 400 µl of fluorescein isothiocyanate (FITC)/rhodamine dextran (40 µg/mL FITC-labelled 4 kDa dextran, and 40 µg/mL rhodamine labelled 70 kDa dextran in Hank’s Buffered Salt Solution). A ligature was applied as the tube was removed. The length and width of the segment was measured and then submerged in 10 mL of HBSS maintained at 37 °C and percolated with carbogen (95% O₂, 5% CO₂). Samples (100 µl) were collected from surrounding media at baseline and every ten minutes for thirty minutes. Fluorescence was measured using a SpectraMax M3 spectrophotometer (Molecular Devices, USA) fluorescence at 438ex/544em for FITC and once at 520ex/590em for rhodamine. A leakage ratio was calculated for each time point by normalizing dextran (µg) to the gut volume then dividing the 4 kDa dextran by 70 kDa dextran to obtain a ratio. Serum LPS measurement was carried out using the Pyrochrome Limulus Amoebocyte Lysate (LAL) assay according to manufacturer’s instructions (Associates of Cape Cod Incorporated).