Model 680xr microplate reader

The wet weight of the samples was weighed before performing analysis.
DNA was isolated using a QIAamp DNA mini Kit (Qiagen) following the manufacturer's protocol. DNA content was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific; N = 11) and corrected for the corresponding wet weight of the measured sample (ng DNA/mg wet weight tissue). The quality and length of DNA base pairs (BP) was measured using a 2100 BioAnalyzer (Agilent technologies) using a DNA‐1000 kit (Agilent Technologies).
Total collagen content of the samples was determined using a Total Collagen Kit (Quickzyme Biosciences, N = 9). Collagen content was measured in a clear 96‐well plate at 570 nm using an Omega POLARstar Microplate reader (BMG labtech). The content was corrected for the wet weight of the corresponding samples (µg Collagen/mg wet weight tissue).
Sulfated glycosaminoglycan (sGAG) was determined using a Blyscan glycosaminoglycan assay (Biocolor; N = 15). Samples were digested in a Papain (Sigma) solution (10 mg/ml) at 65°C for 8 h. sGAG was isolated from the sample digest according the manufacturers protocol. The sGAG content was measured by absorbance measurements (680 nm) in a clear 96‐well plate using a model 680 XR microplate reader (Bio‐Rad).

Total phosphate content in starch was determined essentially according to the method of Morrison [39 (link)] with some modifications. About 20 mg of dry starch were suspended in 250 μl of 70% (w/w) HClO₄ and completely charred at 250°C for 25 min. The solution was clarified by adding 50 μl 30% (w/v) H₂O₂ and gently boiled for 2 min. Once the solution had cooled, water was added to a final volume of 2 ml and 100 μl of the sample was transferred into a 96-well microtiter plate, followed by adding 200 μl of colour reagent [0.75% (w/v) (NH₄)₆M_O7O₂₄.4H₂O, 3% (w/v) FeSO₄.7H₂O and 0.75% (w/v) SDS dissolved in 0.375 M H₂SO₄]. Absorbance at 750 nm was then measured in a Model 680 XR Microplate Reader (Bio-Rad, US), and a calibration curve was used to calculate the concentration in nmol PO₄/mg starch.

CSF total antioxidant capacity was measured using a standardized commercial kit (Antioxidant Assay Kit, Cayman Chemical Company, Ann Arbor, MI USA). This assay relies on the ability of antioxidants to inhibit oxidation of 2,2′-Azino-di-[3-ethylbenzthiazoline sulphonate] (ABTS) by metmyoglobin. Briefly, 10 µL of metmyoglobin was added to 10 µL of diluted sample. 150 µL of chromagen, containing ABTS, was subsequently added and the reaction initiated by adding 40 µL of 441 µM hydrogen peroxide. The plate was incubated on a shaker for 5 minutes and the amount of oxidized ABTS was measured spectrophotometrically, at an absorbance of 750 nm, using a Model 680XR microplate reader (BioRad, Hercules).

Heterologous expression, purification, and biochemical characterization of yeast Aad proteins were carried out in parallel, with the PcAad1p serving as a reference. PcAad1p and all recombinant yeast Aad proteins bear an N-terminal His₆-GST and a C-terminal His₆ tag and were purified following the GST-affinity batch purification (10 (link)). The activities of purified reference PcAad1p, yeast Aadp proteins, and mutated yeast Aad recombinant proteins were assayed against a panel of aliphatic/aromatic aldehydes and aryl-alcohols using both NAD(P)H and NAD(P)⁺ as reduction and oxidation cofactors (10 (link)). Reactions were quantified spectrophotometrically by following the consumption or production of cofactor NAD(P)⁺(H) at λ = 340 nm (ε₃₄₀ = 6.2 mM⁻¹ · cm⁻¹) in 250-μl microplates containing 0.3 mM cofactor and 0.3 mM substrates (10 (link)) (microplate reader model 680XR; Bio-Rad). Kinetic parameters (K_m and V_max) quantifying NADP⁺(H) consumption/formation were assayed at 355 nm (ε₃₅₅ = 5.12 mM⁻¹ · cm⁻¹) using a UV-visible spectrophotometer (Shimadzu UV1800) in 1-ml cuvettes, as described previously; the substrate absorption at this wavelength is negligible (10 (link)). For each recombinant protein, significant differences relative to a pGS-21a blank plasmid were calculated using a 2-tailed t test at a P value of <0.05.