Trichloracetic acid

Dimethylformamide (DMF), trichloracetic acid (TCA), horseradish peroxidase (HRP) (type VI, ≥250 units/mg), catalase (from bovine liver, 2,000–5,000 units/mg), 4-aminoantipyrine (4-AP), phenol, graphene oxide (GO), activated charcoal, agar powder, potassium chloride, silver nitrate, potassium hexachloroplatinate (IV), gold(III) chloride trihydrate, aceton, 2-isopropanol, ethanol, and other chemicals were purchased from Sigma-Aldrich (St. Louis, United States). All reagents were of analytical grade. Mn(III) meso-tetra(N-methyl-4-pyridyl) porphine pentachloride (MnTMPyP) and Mn(III) meso-tetra(4-pyridyl) porphine chloride (MnTPyP) (>95%) were obtained from Frontiers Scientific Inc. Phosphate buffer (PB) solutions (0.1 M, pH 7.4) were prepared from disodium hydrogen phosphate and sodium dihydrogen phosphate. The pH of the solutions was controlled by the pH-meter 765 (Knick GmbH). All solutions were prepared using a Milli-Q water purification system (Merk KGaA, Germany).

For analysis of lipid peroxidation, MDA leaf material (0.5 g) was homogenized using 5% trichloracetic acid (TCA) (Sigma-Aldrich). The method of Hodges et al. with slight modifications was used to estimate MDA. The homogenates were centrifuged at 13 g for 17 min (centrifuge MPW-351 R) and the supernatant was added to 20% TCA containing 0.5% thiobarbituric acid (TBA) (Alfa Aesar). The homogenate was incubated in a heater at 95 °C for 30 min (Blockthermostat BT 200, Kleinfeld, Labortechnik) and subsequently cooled on ice. The optical density was measured at 532 and 660 nm using a spectrophotometer (Analytik Jena Specord 210 Plus, Analytik Jena, Jena, Germany)). The results were expressed in µmol g⁻¹ FW [63 (link)].

Caffeine (CAF), Cytotoxicity Detection Kit (LDH) Roche, D-mannitol (MAN), D-Mannitol Colorimetric Assay Kit, disodium fumarate, deoxynivalenol (DON), L-glutamate, lucifer yellow (LY), and sodium pyruvate were obtained from Sigma Aldrich (St. Louis, MO, USA). All inorganic salts required for preparing Krebs Bicarbonate Buffer (KRB), ethanol, and glucose were purchased from Avantor (Gliwice, Poland). Amoxicillin trihydrate (AMX) and doxycycline hyclate (DOX) were generously donated by the pharmaceutical company Biofaktor Sp. z o.o. (Skierniewice, Poland) The quality of antibiotics was consistent with the monographs of the European Pharmacopoeia and corresponded to the quality of active substances used in the production of veterinary drugs.
Tissue transportation, preparation, and incubation were performed in Krebs Bicarbonate Buffer (KRB) containing 108 mM NaCl, 4.7 mM KCl, 1.8 mM Na₂HPO₄, 0.4 mM KH₂PO₄, 15 mM NaHCO₃, 1.2 mM MgSO₄, 1.25 mM CaCl₂, 11.5 mM glucose, 4.9 mM L-glutamate, 5.4 mM disodium furmate, and 4.9 mM sodium pyruvate at pH 7.4, and saturated with oxygen using a 95%/5% O₂/CO₂ mixture by gassing for 60 min [37 (link)].
For HPLC and LC-MS/MS analysis, acetonitrile, methanol, and formic acid (HPLC grade) were obtained from Avantor Chemicals (Radnor, PA, USA), trichloracetic acid and heptafluorobutyric acid were obtained from Sigma Aldrich (St. Louis, MO, USA).

After cells (2 × 10⁵) where indicated were washed with cold PBS three times, sample preparation and intracellular NAD measurement were carried out as described in specification of NAD/NADH Quantification Colorimetric Kit (BioVision). The sample preparation^{40 (link)} for LC-MS analysis^{41 (link)} of quinolinic acid (QA) was performed as previously described. Briefly, 10 μM deuterium D3 quinolinic acid (QA-d3, J&K Scientific Ltd) was spiked to the culture supernatants as the internal standard. Proteins in supernatants were precipitated by trichloracetic acid (Sigma, 3% final concentration), the supernatant was then filtered through a spin column (MW cutoff = 3 kDa, Millipore Amicon) at 14,000 × g at 4 °C for 10 min. The filtrates were 10 × diluted and injected (5 µL) to a Acquity H-Class UPLC system (Waters, HSS T3 2.1 × 100 mm, 1.8 µm particle column. Solution A, 0.3% formic acid in water, and solution B, 0.3% methanol in water). The analytes were eluted (0.3 mL/min flow rate) by the following gradients: 0–1.7 min (95%A and 5%B), 5.0–6.0 min (50% A and 50%B) and 6.5–9.0 min (95%A–95%B). The triple quadrupole in the + ve ESI mode was used in a Quattro Premier XE mass spectrometer (Waters, MA). Standard solutions of QA for calibration curves were prepared from stock solutions. The final concentrations of unknowns were calculated by interpolation of the standard curves.

For analysis of MDA and H₂O₂, leaf material (0.5 g) was homogenized using 5% trichloracetic acid (TCA) (Sigma-Aldrich, St. Louis, MO, USA). The method of Hodges et al. [72 (link)] with slight modifications, have been used to estimate MDA. The homogenates were centrifuged at 12.13× g for 17 min (centrifuge MPW-351 R), and supernatant was added to 20% TCA containing 0.50% thiobarbituric acid (TBA) (Alfa Aesar, Haverhill, MA, USA). The homogenate was incubated in a heater at 95 °C for 30 min (Blockthermostat BT 200) and then subsequently cooled on the ice. The optical density was measured at 532 and 660 nm by spectrophotometer (Analytik Jena Specord 210 Plus, Analytik Jena, Jena, Germany). The results were expressed in µmol g⁻¹ FW [72 (link)]. To avoid desiccation effects, the RWC was used as a factor to calculate the MDA content and to estimate the effective differences between treatments.

The content of MDA in each reaction mixture was quantified as described by Vasantha Rupasinghe and Yasmin [9 (link)], with some modifications. The thiobarbituric acid (TBA) reagent (20% (w/v) trichloracetic acid (Sigma Aldrich) along with 0.375% (w/v) TBA in 0.25M HCl (Sigma Aldrich)) was added to each reaction mixture. The mixtures were incubated for 30 min at 95 °C and then cooled to room temperature. The MDA-TBA adduct which formed was extracted from the mixture with an equal volume of butanol (Sigma Aldrich). The extraction was done in two steps: The mixtures were vigorously vortexed for 15 min, and then centrifuged for 5 min at 16,000× g. Each butanol fraction was transferred to a 96-well plate, and the absorbance was measured at 532 nm using a FluoStar Omega spectrophotometer (BMG Labtech, Ortenberg, Germany).