Powerwave ht

Total ATP levels were monitored using a CellTiter-Glo Luminescent Cell Viability Assay as per the manufacturer's instructions (G7572, Promega, Durham, NC, USA). CellTiter-Glo was added to 1 × 10⁶ cells and placed on an orbital shaker to induce cell lysis, and then the samples were read on a chemiluminescence plate reader (VICTOR light 1420; integration time of 1 s). Total NADH and NAD levels were measured using the NAD/NADH Quantitation Colorimetric Kit (K337-100, BioVision, Milpitas, CA, USA). To obtain total NAD (NADH and NAD), NADH/NAD Extraction Buffer was added to 2 × 10⁵ cells and a portion of NADH/NAD extract was heated for 30 min at 60°C to decompose NAD. The NADH and NAD extracts were converted to NADH using the NADH cycling enzyme and then read on an ELISA reader (Power wave HT, Biotek, Winooski, VT, USA).

Immunoreactivity of pin-bound synthesized epitopes was tested at RT by ELISA [51 (link),53 (link)] against three groups of pooled sera: initial CDI episode, umbilical cord blood, or healthy volunteers. The assay consisted of four main steps: pins incubation with: (1) blocking solution containing 1% BSA (SeraCare, Milford, MA, USA) in TBS-T; (2) primary antibodies in a 1:1000 dilution in TBS-T with 0.1% BSA in TBS-T; (3) secondary antibodies – anti-human IgG conjugated with AP (cat. A1543-1ml, Sigma-Aldrich) in a 1:10000 dilution in TBS-T; and (4) Alkaline Phosphatase Yellow Liquid Substrate System for ELISA (AP Yellow, Sigma-Aldrich). Plates were read at 405 nm (PowerWave HT, BioTek Instruments, Winooski, VT, USA). After assay, bound antibodies were stripped off by sonication in disruption buffer (1% sodium dodecyl sulfate, 0.1% 2-mercaptoethanol and 0.1 M Na₃PO₄) preheated to 60 °C, washed in water and methanol, and left to dry.
Based on the results obtained in ELISA assays, the baseline was calculated, and peptides were selected for further analysis. In his work, Carter suggested that the background should be calculated as the mean of the 10–25% of the lowest results [51 (link)]. We, however, considered the baseline as a mean of the results obtained for all peptides, which is a more restrictive approach.

The T-cell lymphoma (Jurkat) and B-cell lymphoma (Ramos) were cultured using RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin. An initial cell densities of 5 × 10³ cells/well was uniformly maintained in a 96 well plate. The B16-F1 melanoma cells were grown in DMEM medium containing 10% FCS, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin and 55 μM 2-mercaptoethanol. The cells were trypsinized and plated in a 96-well plate at a density of 5 × 10³ cells/well. The cells were grown overnight at 37 °C using a 5% CO₂ incubator. The cells were treated with different peptide concentration followed by cell proliferation assay. The MTT assay kit (Cayman chemicals, USA) was used to measure the cell viability at a peptide concentration varying from 2 to 15 μM (in triplicate). Fresh culture medium and cells in medium without peptide were considered as negative and positive controls for viability assay and was monitored at 24, 48 and 72 h. The Cayman MTT cell proliferation assay protocol was followed, and the absorbance were recorded at 570 nm using a microplate reader (PowerWaveHT, BioTek Instruments, USA).

Our coupled activity assay combines 2 different enzyme activities to detect methylselenol production, i.e., thioredoxin reductase 1 and KYAT1. Briefly, 100 µL of reaction mixture contained 100 mM potassium phosphate buffer pH 7.4, 1–10 mM of MSC, 100 µM of αKG, 100 µM of KMB, 10 µM of PLP, 0.4 µg mammalian TrxR1, 400 µM of NADPH and 50 ng to 400 ng of pure KYAT1 protein or 20 µg of protein from whole cell lysate. The reaction mixture was pre-incubated for 5 min at 37 °C before adding TrxR1 and NADPH. Continuous measurement of NADPH consumption was recorded at 340 nm for every 30 s using a spectrophotometer (PowerWave HT, BioTek). The assay mixture without TrxR1 was used as blank. Under this condition, the molar extinction coefficient was 6220 M⁻¹ cm⁻¹ [19 (link),20 (link)] for NADPH. T_max represents the time taken to consume all the available NADPH in the assay mixture. To assess the inhibitory effects of β-elimination inhibitors on the enzyme activity, the respective compounds were added at the desired concentration as indicated with 100 ng of KYAT1 enzyme.

KYAT1 transamination activity was measured with pure protein or whole-cell lysate according to the published procedure [15 (link),16 (link)]. Briefly, 50 µL of the reaction mixture contained 20 mM of l-Phe, 5 mM of KMB, 100 mM of ammediol-HCl buffer (pH 9.0) and 50 ng to 400 ng of pure KYAT1 protein or 20 µg of protein from whole-cell lysate. The assay mixture was incubated at 37°C and the reaction was terminated by adding 150 µL of 1 M NaOH at different time points. The production of phenylpyruvate-enol was assessed by measuring the absorbance at 320 nm in a spectrophotometer (PowerWave HT, BioTek, Winooski, VT, USA). The assay mixture without enzyme was used as blank. Under this condition, the molar extinction coefficient is 16,000 M⁻¹ cm⁻¹ for phenylpyruvate-enol [15 (link)]. UV flat-bottom plates (Corning, Kennebunk, ME, USA) were used for this assay. To assess the effect of transamination inhibitors on the enzyme activity, the respective compounds were added at the desired concentration as indicated with 50 ng of KYAT1 enzyme. The reaction was pre-incubated for 5 min at 37 °C before being added the enzyme and KMB.

The cell viability for all the concentrations of the aqueous extract tested was verified by MTT assay on HEK293 and R3/1-Nf-κB cells in transparent 96-well plates seeded with 10,000 and 5000 cells/well, respectively, in Dulbecco’s modified Eagle medium (DMEM; Lonza, Verviers, Belgium) supplemented with 10% fetal bovine serum (FBS; Gibco, Gaithersburg, MD, USA) and 1% Penicillin/Streptomycin (Lonza). After 18 h incubation with the extract at the appropriate concentrations (1, 10, 50, and 100 µg/mL), media were removed and, for the R3/1-NF-κB cell line, one wash with 100 µL PBS occurred. Subsequently, 50 µL/well of DMEM was added, followed by 11 µL/well MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, purchased from Merck KGaA, Darmstadt, Germany) reagent (5 mg/mL). After 4 h incubation, DMEM was removed, cells were treated with lysis buffer (100 µL/well) (HCl 8 mM, 5% TWEEN20, DMSO), and the 96-well plate was shaken for 15 min in a plate shaker in the dark. Absorbance at 575 nm and 630 nm was measured using a plate reader (BioTek’s PowerWave HT, Winooski, VT, USA). Cells incubated with media were used as a control of 100% proliferation, while cells incubated with DMSO (4%) were used as a negative control.

MnSOD activity was measured using a commercially available assay kit (Sigma-Aldrich, St. Louis, MO, USA). Muscle was homogenized in buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA and 1 mM DTT) and centrifuged at 10,000× g for 15 min at 4 °C. The resulting supernatant (20 µL) was added to 200 µL of Working Solution and 20 µL of Enzyme Working Solution (Sample) or 200 µL of WST Working Solution and 20 µL of dilution buffer (Blank 2). Super clean H₂O (20 µL) was added to 200 µL of WST working solution and 20 µL Enzyme Working Solution (Blank 1) or 20 µL dilution buffer (Blank 3). After 20 min incubation at RT, samples were read at 450 nm using a spectrophotometer (Powerwave HT, BioTek Instruments Inc.). MnSOD activity was assessed by pre-incubating the homogenate with potassium cyanide. Activity (units/mg protein) was calculated using the following equation:
where A = absorbance