Versamax spectrophotometer

Neuropeptide Y (NPY) was measured using a commercially available ELISA kit (EZHNPY-25K, Millipore, Temecula, CA, USA), according to the manufacturer’s instructions. Briefly, this assay is a sandwich ELISA based on capture of NPY by human NPY IgG precoated on wells. Thereafter, a secondary biotinylated antibody is binding to NPY after brief washing, followed by conjugation of horseradish peroxidase to the immobilized biotinylated antibodies after further washing. Finally, 3,3′,5,5′-tetra-methylbenzidine substrate is added before measurement of absorbance at 450 nm, corrected from the absorbency at 590 nm (VersaMax spectrophotometer, Molecular Devices, San Jose, CA, USA).

Blood was collected upon euthanization and allowed to coagulate at room temperature before centrifugation; serum was aliquoted and frozen at −80°C. Blood urea nitrogen (BUN) and creatinine levels were measured using commercially available kits (Sigma‐Aldrich, St. Louis, MO) on a VersaMax Spectrophotometer (Molecular Devices, Sunnyvale, CA).

Cytotoxicity assays were conducted in triplicate under the same experimental conditions. Following stimulation, cells were incubated with WST-8 (CCK-8 #311KTA1020, Tebubio, Le Perray, France) and optical density was measured at 450 nm using a VERSAmax spectrophotometer (Molecular Devices, San Jose, CA, USA). WST-8 is a tetrazolium salt that is reduced in a yellow-colored formazan dye by mitochondrial succinate dehydrogenase (an indicator of metabolically active cells). Formazan product formation is directly proportional to the number of living cells and their metabolic activity. The percentage of cell viability was calculated relative to the LPS-stimulated condition.

The pericardium was digested with papain solution at 60°C for 6 h. papain
(Sigma, St. Louis, MO) was dissolved at 400 mg/mL in 0.1 M phosphate buffer
(pH 6.0) with 5 mM cysteine hydrochloride and 5 mM EDTA. The lysates were
used for detection of the sGAG amount. The amount of sGAG was measured using
a Blyscan sGAG assay kit (Biocolor, Newtownabbey, UK) according to the
manufacturer's manual. The tissue lysate was mixed with Blyscan dye to bind
the GAG. The GAG-dye complex was then collected by centrifugation.
Subsequently, the supernatant was removed and the tube drained, and the
dissociation reagent was added. Then solution was transferred into a 96-well
plate. Absorbance against the background control was obtained at a
wavelength of 656 nm on a Versamax spectrophotometer (Molecular Devices,
Sunnyvale, USA). The sGAG amount was calculated based on a standard curve
obtained with the standard sGAG supplied with the kit.

Respiration rate and H₂O₂ production were normalized to total mitochondrial protein content (Bradford assay, VersaMax spectrophotometer: Molecular Devices, Sunnyvale, CA, United States), and H₂O₂ production was also expressed relative to the rate of O₂ consumption. To estimate mitochondrial efficiency, the respiratory-control ratio (RCR) was calculated as OXPHOS_CI/Leak_T,CI, and the OXPHOS coupling-efficiency ratio was calculated as 1-Leak_T,CI/OXPHOS_CI. The OXPHOS-control ratio was calculated as OXPHOS_CI+CII/ET_CI+CII. We also calculated the ratio of phosphorylated ADP to the atoms of consumed O₂ (P:O ratio), by making linear extrapolations of O₂ concentration during OXPHOS (immediately after ADP addition) and Leak_T (immediately after ADP depletion), as described by Illingworth ; the difference in O₂ concentration at the intercepts is the total O₂ uptake. Statistical significances were determined using generalized linear models (GLMs), with sequential Sidak post hoc tests, for pairwise comparisons. Developmental O₂ was the between-group factor, and the pooled mitochondrial samples were the random factors. Data were considered significant when P ≤ 0.05.

TP was fractionated by ion-exchange liquid chromatography. Initially, 200 µg of material were applied to a diethylaminoethyl cellulose anion exchange column (DEAE sepharose) (GE Healthcare) coupled to the FPLC (fast protein liquid chromatography) Äkta Prime equipment (Amersham Biosciences, Amersham, UK, SN: 1,102,380), equilibrated in 0.02 M Tris-HCl buffer, pH 8.0. The GAGs were eluted from the column in a linear NaCl gradient (0–3 M) using a 0.02 M Tris-HCl buffer containing 3 M NaCl, pH 8.0, at a flow rate of 3 mL per minute, with fractions of 3 mL per tube being collected. Next, conductivity was measured to assess the NaCl concentration. Thereby, 240, 370, and 700 mM NaCl concentrations were determined as concentrations used in a stepwise NaCl gradient for bulk sulfated polysaccharide fractionation. The sulfated polysaccharide elution pattern was assessed by metachromasia properties using 100 µL of DMB and 20 µL of sample in a 96-well plate. Then, read in a Versamax spectrophotometer with a microplate reader (Molecular Devices, San Jose, CA, USA) at 525 nm.
Finally, the fractions containing metachromatic material correlated to the peaks in the graph (SP1 and SP2) were grouped and precipitated in 70% ethanol at −20 °C for 24 h. The suspension was centrifuged (3200 rpm for 30 min), the pellet was rinsed in 80% ethanol, centrifuged again (3200 rpm for 30 min), and let air dry.