TRAIL plasma levels were measured using Quantikine solid-phase ELISA Kits according to the manufacturer's instructions (R&D Systems, Minneapolis, MN). The absorbance was recorded (ELx808 microplate reader, Bio-Tek Instruments), and data collection and analysis were performed using the Gen5 software Version 1.08 (Bio-Tek Instruments). Plasma samples were collected in tubes containing EDTA as an anticoagulant. As per the manufacturer, the mean normal TRAIL level in plasma is 82 pg ml−1 (range: 34–163 pg ml−1).
Gen5 software version 1
Manufactured by Agilent Technologies
Sourced in United States
Gen5™ software, version 1.1, is a data analysis and management software designed for Agilent Technologies' lab equipment. It provides functionality for acquiring, analyzing, and managing data generated from Agilent instrumentation.
Automatically generated - may contain errors
Lab products found in correlation
Elx808, by Agilent Technologies (3 mentions)
Synergy ht microplate reader, by Agilent Technologies (2 mentions)
Black 96 well plate, by Thermo Fisher Scientific (2 mentions)
Human cxcl12 elisa kit, by R&D Systems (1 mentions)
Quantikine kits, by R&D Systems (1 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
Synergy 4, by Agilent Technologies (1 mentions)
Trolox, by Roche (1 mentions)
Synergy 2 plate reader, by Agilent Technologies (1 mentions)
9 protocols using gen5 software version 1
1
Quantification of TRAIL Plasma Levels
2
Quantitative CXCL12 Secretion Assay
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Supernatants from each group were collected, and the concentration of CXCL12 was detected using a human CXCL12 ELISA kit (R&D Systems), and the CXCL12 level in the supernatant was measured using a Quantikine kit within 30 minutes according to the manufacturer’s instructions (R&D Systems). High intra-assay and inter-assay precision was established by the manufacturer. The absorbance was recorded using a microplate reader (ELx808, Bio-Tek Instruments), and the data collection and analysis were performed using Gen5 software Version 1.08 (Bio-Tek Instruments).
3
Quantification of CCL3 and CCL4 in Serum Samples
4
Measuring Antioxidant Capacity via ORAC
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Antioxidant activity was determined by the oxygen radical absorbance capacity (ORAC) method as described previously [24 (link)]. Briefly, the reaction was performed at 37 °C in 75 mM phosphate buffer at pH 7.4. The reaction mixture (200 μL) contained 180 μL of 70 nM fluorescein, 90 μL of 12 mM 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) and 30 μL of a diluted sample or the standard 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) at concentrations ranging from 1 to 160 μM. Reaction mixtures were placed in a black 96-well plate, and the fluorescence was read in a Synergy HT microplate reader (BioTek Instruments, Winooski, VT, USA) every minute at excitation and emission wavelengths of 485 and 520 nm, respectively. The equipment was controlled by Gen5™ software, version 1.1 (BioTek Instruments, Winooski, VT, USA). The results were expressed as μmol Trolox equivalents (TE)/100 g dw.
5
Antioxidant Activity Evaluation by ORAC
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The antioxidant activity was determined by the ORAC method as described previously [21 (link)]. Briefly, the reaction was performed at 37 °C in 75 mM phosphate buffer at pH 7.4. The reaction mixture (200 μL) contained 180 μL of 70 nM fluorescein, 90 μL of 12 mM AAPH and 30 μL of diluted sample or the standard Trolox at concentrations ranging from 8 to 160 μM. Reaction mixtures were placed in a black 96-well plate (Fisher Scientific, Waltham, MA, USA) and the fluorescence was read in a Synergy HT microplate reader (BioTek Instruments, Winooski, VT, USA) every minute at excitation and emission wavelengths of 485 and 520 nm, respectively. The equipment was controlled by Gen5™ software, version 1.1 (BioTek Instruments, Winooski, VT, USA). Quantification of ORAC was made according to the linear calibration curves of standard Trolox (r2 > 0.99). Results were expressed as µmol Trolox equivalents (TE) per 100 g dw.
6
Fluorescent HDAC Activity Assay
7
Oxygen Radical Absorbance Capacity Assay
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The AA was determined by the oxygen radical absorbance capacity (ORAC) method described previously [15 (link)]. Briefly, the reaction was performed at 37 °C in 75 mM phosphate buffer at pH 7.4. The reaction mixture (200 μL) contained 180 μL of 70 nM fluorescein, 90 μL of 12 mM 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) and 30 μL of diluted sample or standard (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, Trolox, Hoffman-LaRoche, basel, Switzerland) at concentrations ranging from 1 to 160 μM. Reaction mixtures were placed in a black 96-well plate (Fisher Scientific, Hampton, VA, USA) and the fluorescence was read in a Synergy HT microplate reader (BioTek Instruments, Winowski, VT, USA) every minute at excitation and emission wavelengths of 485 and 520 nm, respectively. The equipment was controlled by Gen5™ software, version 1.1 (BioTek Instruments). Results were expressed as μmol Trolox equivalents (TE)/g dw.
8
Antioxidant Capacity Determination by ORAC
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The ORAC method was previously described by Abdel-Aal and Rabalski [24 (link)]. A total of 25 μL of sample extract, Trolox standard solution (0–140 μg/mL), or nano pure water (blank) were mixed with 150 μL fluorescein in each of the 96 micro-plate well. The mixture was conditioned at 37 °C for 15 min, then 25 μL of 2,2′-Azobis (2-methylpropion-amidine) dihydrochloride (AAPH) as a peroxyl radical generator was added to start the decaying of fluorescein. The degradation of fluorescein progressed for 60 min in the heated chamber of BioTech Synergy H4 with the following settings: fluorescence excitation at 485 nm, emission wavelength 528 nm, and reading was taken every min for 60 min. The microplate fluorescent reader was operated by Gen 5 software version 1.11.5 (BioTek). The data are presented as means of ORAC values in μmol Trolox equivalent/g sample dry weight.
9
Quantifying Cell-Derived Cytokines
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Concentrations of IL-6, tumor necrosis factor α (TNF-α), and IL-1β were determined in cell culture supernatants via ABTS-based enzyme-linked immunosorbent assay (Peprotech; #900-K16, #900-K25, and #900-K95). Cell culture supernatants were measured undiluted or diluted 1:50 with PBS. Assays were performed as recommended by the manufacturer. Absorbance was measured on a BioTek Synergy 2 plate reader, and cytokine concentrations were calculated via BioTek Gen5 software version 1.11.5.
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