The antioxidant capacity of umbrian LBB extract was determined using the ORAC method as previously reported [40 (link)]. We have chosen ORAC method because it is a robust and reliable method. In fact, the ORAC assay, other common measures of antioxidant capacity include ferric ion reducing antioxidant power and trolox equivalence antioxidant capacity assays and therefore is considered to be a preferable method because of its biologic relevance [41 ]. A duplicate extraction was performed for each sample and used to evaluate the lipophilic (L-ORACFL) and hydrophilic ORACFL (H-ORACFL) values. Evaluations of the lipophilic and hydrophilic ORACFL in the LBBs samples were performed separately, and the total antioxidant capacity (TAC) was calculated by adding the L-ORACFL and H-ORACFL values. The ORACFL assays were carried out on a FLUOstar OPTIMA microplate fluorescence reader (BMG LABTECH, Offenburg, Germany) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. The procedure was based on the method of Zulueta et al. (2009) with slight modifications. Briefly, 2,20-azobis (2-methylpropionamide) dihydrochloride (AAPH) was used as a peroxyl radical generator, trolox was used as a reference antioxidant standard, and fluorescein was used as a fluorescent probe. The data are expressed as micromoles of trolox equivalents (TE) per gram of sample (μmol TE/g).
Fluostar optima microplate fluorescence reader
Manufactured by BMG Labtech
Sourced in Germany
The FLUOstar OPTIMA is a microplate fluorescence reader manufactured by BMG LABTECH. It is designed to measure fluorescence in microplates. The FLUOstar OPTIMA provides basic fluorescence detection capabilities for microplate-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Trolox, by Merck Group (3 mentions)
Amplex ultrared, by Thermo Fisher Scientific (1 mentions)
Amplex ultrared, by BMG Labtech (1 mentions)
α β integrin mediated cell adhesion array combo kit, by Merck Group (1 mentions)
Flat bottomed 96 well plate, by Thermo Fisher Scientific (1 mentions)
Dual luciferase reporter assay, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
15 protocols using fluostar optima microplate fluorescence reader
1
Antioxidant Capacity of Umbrian LBB Extract
2
ORAC Assay for Antioxidant Capacity
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The antioxidant capacity of all compounds was determined using the ORAC method. Hydrophilic extraction was performed on two independent occasions for each sample. The procedure was based on the method of Zulueta et al. [23 (link)], with slight modifications [24 (link)]. Briefly, 2,20-azobis (2-methylpropionamide) dihydrochloride (AAPH) was used as a peroxyl radical generator, fluorescein was used as a fluorescent probe and Trolox was used as a reference antioxidant standard. The data are expressed as micromoles of Trolox equivalents (TE) per gram of sample (μmol TE/g). The ORAC assay was carried out on a FLUOstar OPTIMA microplate fluorescence reader (BMG LABTECH, Offenburg, Germany) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm.
3
Measuring Extracellular H2O2 in Cells
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Extracellular H2O2 was measured in intact cells using horseradish peroxidase-linked Amplex Ultra Red (Invitrogen, Carlsbad, CA, USA). Briefly, Amplex Ultra Red (50 μM for HLE cells and 100 μM for Huh7 cells) and horseradish peroxidase (0.1 U/ml for HLE cells and 0.2 U/ml Huh7 cells) were added to the cellular samples for 2 h. Fluorescence readings were made at the times indicated in the corresponding figure, in duplicate in a 96-well plate at ex/em 530/590 nm using 100 μl samples of medium. Fluorescence was measured in a Fluostar Optima microplate fluorescence reader (from BMG LABTECH, Ortenberg, Germany) and expressed as percentage of control after correction for cell number (crystal violet assay) with Amplex Ultra Red.
4
Antioxidant Capacity of Umbrian LBB Extract
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The antioxidant capacity of Umbrian LBB extract was determined using the ORAC method [18 (link)]. The hydrophilic and lipophilic fractions were extracted according to Prior et al. [19 (link)]. A duplicate extraction was performed for each sample and used to evaluate the lipophilic (L-ORACFL) and hydrophilic ORACFL (H-ORACFL) values [19 (link)]. Evaluations of the lipophilic and hydrophilic ORACFL in the LBBs samples were performed separately, and the total antioxidant capacity (TAC) was calculated by adding the L-ORACFL and H-ORACFL values [20 (link)]. The ORACFL assays were carried out on a FLUOstar OPTIMA microplate fluorescence reader (BMG LABTECH, Offenburg, Germany) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. The procedure was based on the method of Zulueta et al. [21 (link)] with slight modifications. Briefly, 2,20-azobis (2-methylpropionamide) dihydrochloride (AAPH) was used as a peroxyl radical generator, Trolox was used as a reference antioxidant standard, and fluorescein was used as a fluorescent probe. The data are expressed as micromoles of Trolox equivalents (TE) per gram of sample (μmol TE/g).
5
Muscle Antioxidant Capacity Evaluation
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The antioxidant capacity was evaluated using the oxygen radical absorbance capacity (ORACFL) method. One gram of muscle was mixed with a buffer containing KH2PO4 13.19 g/L and K2HPO4 10.26 g/L (v/v) solution at pH 7.2, homogenized with Ultra-Turrax homogenizer (Ultra Turrax T25 Basic, IKA Labortechnik Janke & Kunkel GmbH, Staufen, Germany) for 1 minute, and then vortexed for 2 minutes. The homogenates were centrifuged at 6000 rpm at 4°C for 20 minutes, and the supernatant was used for the determination of the antioxidant capacity using the ORACFL method based on the fluorescence decay rate of a probe in the presence of a radical oxygen species compared with that of a reference standard, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, Sigma-Aldrich, Steinheim, Germany). The ORACFL assays were carried out on a FLUO-star OPTIMA microplate fluorescence reader (BMGLABTECH, Offenburg, Germany) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm, as reported in Branciari et al. (2015 ). The results were expressed as µg of Trolox equivalents per 100 g sample.
6
Antioxidant Capacity of Polyphenolic Extracts
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The antioxidant capacity of polyphenolic extracts was measured using the Oxygen Radical Absorbance Capacity method (ORACFL). This test is based on the decay rate of the fluorescence probe due to radical oxygen species (ROO) compared to the reference standard, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, Sigma-Aldrich, Steinheim, Germany). One mL of LE or 1 g of EE was mixed with a buffer, 75 mM, at pH 7.2, containing 13.19 g of K2HPO4 and 10.26 g of KH2PO4 in 900 mL of deionized water. The obtained mixture was homogenized for 1 min with an Ultra-Turrax homogenizer (Ultra Turrax T25 Basic, IKA Labortechnik Janke & Kunkel GmbH, Stavfen, Germany), and then vortexed for 2 min. After centrifugation at 6000 rpm for 20 min at +4 °C, the supernatant was used for antioxidant capacity determination. ORACFL assays were executed on an FLUO-star OPTIMA microplate fluorescence reader (BMGLABTECH, Offenburg, Germany) at excitation and emission wavelengths of 485 and 520 nm, respectively. The results of the test are expressed as µg of Trolox equivalents (TE) per g of sample.
7
Antioxidant Capacity Evaluation in Burgers
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The antioxidant capacity was evaluated for the synthetic and natural additives and burgers over the course of their shelf-life using the oxygen radical absorbance capacity method (ORACFL). One gram each of CM, PE and burger samples was separately mixed with a buffer, 75 mM, pH 7.2, containing 13.19 g of K2HPO4 and 10.26 g of KH2PO4 in 900 mL of deionized water, homogenized with an Ultra-Turrax homogenizer (Ultra Turrax T25 Basic, IKA Labortechnik Janke & Kunkel GmbH, Stavfen, Germany) for 1 min, and then vortexed for 2 min. The homogenates were centrifuged at 6000 rpm at 4 °C for 20 min, and the supernatant was used for the determination of the antioxidant capacity using the oxygen radical absorbance capacity method (ORACFL) based on the fluorescence decay rate of a probe in the presence of a radical oxygen species (ROO) and compared with that of a reference standard, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, Sigma-Aldrich, Steinheim, Germany). The ORACFL assays were carried out on a FLUO-star OPTIMA microplate fluorescence reader (BMGLABTECH, Offenburg, Germany) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. The results are expressed as µg of Trolox equivalents (TE) per 100 g sample.
8
Quantifying Intracellular ROS Levels
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The oxidation-sensitive fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA; from Invitrogen, Carlsbad, CA, USA) was used to analyze the total intracellular content of ROS as previously described.39 (link) Fluorescence was measured in a Microplate Fluorescence Reader Fluostar Optima (BMG LABTECH, Ortenberg, Germany) and expressed as percentage of control after correction with protein content.
9
Quantifying Cell Adhesion to Integrins
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The α/β-Integrin-Mediated Cell Adhesion Array Combo Kit (ECM532, Merck Millipore, Billerica, MA, USA) was used according to the manufacturer's instructions. Each well containing mouse anti-alpha or anti-beta integrin received 100 μl containing 1.5 × 105 cells, as did the bovine serum albumin-coated negative control wells. The plate was incubated for 2 h at 37 °C in 5% CO2 and washed with assay buffer. Cells were then stained with a Cell Stain Solution (provided in kit), incubated for 5 min and washed with dH2O. Extraction buffer (100 μl) was added to each well and left to shake for 5–10 min. Fluorescence was measured in Microplate Fluorescence Reader Fluostar optima (BMG Labtech GmbH, Ortenberg, Germany).
10
pH-Dependent Fluorescence of Multifunctional Nanoparticles
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The fluorescence of MSN/COOH/TAT-FITC/Cit/YSA-BHQ1 under different pH conditions was observed with a fluorescence microscope (Shanghai Bimu Instrument Co. Ltd, China). Briefly, 1 mg MSN/COOH/TAT-FITC/Cit/YSA-BHQ1 was dissolved in 5 mL PBS buffer (pH 7.4). Then the solution was equally divided into four tubes and the pHs were adjusted to 5.5, 6.5, 7.4, and 8.0, separately. For comparison, 0.2 mg MSN/COOH/TAT-FITC or MSN/COOH/TAT-FITC/Cit was dissolved in 1 mL PBS buffer (pH 7.4). Then, the fluorescence of each solution was observed with the fluorescence microscope. The fluorescence intensities of the solutions were measured with a Fluostar Optima fluorescence microplate reader (BMG Labtech GmbH, Ortenberg, Germany) at excitation and emission wavelengths of 490 nm and 530 nm, respectively.
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