Cells of different groups were seeded at 50% confluency in a 24-well plate 16 hrs prior to transient transfection with the DICER1 luciferase reporter and basic-pGL3 luciferase reporter with mutant sites using FuGENE 6 Reagent (Roche, Indianapolis, IN, USA). Luciferase assays were performed at room temperature using an Autolumat LB 953 (Berthold Technologies, Oak Ridge, TN, USA) 48 hrs post-transfection and were normalized with β-gal results, as previously reported 18 (link).
Autolumat lb 953
Manufactured by Berthold Technologies
Sourced in Germany, United States
The AutoLumat LB 953 is a microplate luminometer designed for the detection and quantification of luminescent signals. It is capable of measuring light emission from a variety of luminescent assays, including luciferase, aequorin, and other bioluminescent reporter systems. The instrument features automated plate handling, temperature control, and a high-sensitivity photomultiplier tube detector to provide reliable and accurate luminescence measurements.
Automatically generated - may contain errors
16 protocols using autolumat lb 953
1
DICER1 Luciferase Assay Protocol
2
Transcriptional activity of SET
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The protocol was performed as previously described [56 (link)]. Briefly, HEK293 cells were transfected with SET pcDNA3.1 and pGL3-NFκB-Luc or pGL3-STAT3-Luc (kindly provided by Dr. Squarize) [56 (link)] promoter reporter constructs containing firefly luciferase cDNA, and pRL-null normalization construct containing Renilla luciferase from Renilla reniformis. Firefly and Renilla luciferase activity were measured in the cellular extract using Dual-Glo® Luciferase Assay System (Promega, Madison, WI, USA), 24 h after transfection in a luminometer (AutoLumat LB 953, Berthold).
3
Quantifying NADPH Oxidase Activity
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A lucigenin-enhanced chemiluminescence assay was used to determine NADPH oxidase activity in aortic segments and VSMCs. Arterial segments from the same animal and VSMCs from the same batch were incubated or not with ET-1, previously treated or not with pioglitazone, lactacystin or SP600125. Aortic segments or VSMCs were homogenized in lysis buffer (in mM: 50 KH2PO4, 1 EGTA and 150 sucrose, pH 7.4) on ice to avoid degradation. Protein concentration was determined by using the Bradford reactive and 50 μl of the homogenate (containing 50 µg protein in VSMC and 75–100 µg protein in aortic segments) were transferred to a 96 well plate together with 175 μl of assay phosphate buffer and lucigenin (5 µM); the assay was performed by duplicate. Basal luminescence was measured every 1.8 seconds during 3 minutes in a plate luminometer (Auto-Lumat LB 953, Berthold Technologies, Bad Wildbad, Germany). The reaction was started by the addition of NADPH (0.1 mM) and luminescence was measure every 1.8 seconds during 3 minutes. The buffer blank was subtracted from each reading. Activity was expressed as relative light units per μg of protein. Variations of luminescence were calculated as the amount relative to control or ET-1-treated arterial segments from the same animal or VSMCs from the same batch.
4
Neutrophil Respiratory Burst Assay
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CImax was assessed based on an in vitro reaction between peripheral neutrophils and endotoxin. An endotoxin activity assay was performed as described previously [21 (link)]. Fifty microliter samples of whole blood and appropriate controls were incubated in duplicate with saturating concentrations of an anti-lipid A immunoglobulin M antibody, and then stimulated with opsonized zymosan. The resulting respiratory burst was detected by a chemiluminometer (Autolumat LB953; Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany) as light released from the lumiphore luminol. The maximum stimulated response (termed as CImax by Kiguchi et al. [18 (link)]) was measured using lipopolysaccharide (4.6 ng/mL) as the stimulant.
5
Quantifying Luciferase Activity in Cells
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Cells were washed with PBS and frozen at −80°C. Luciferase activity was measured with an AutoLumat LB953 luminometer (Berthold Technologies, Bad Wildbad, Germany) using a commercially available assay system (E1501; Promega, Madison, WI, USA) following the manufacturer's instructions. All treatments were performed in triplicate. Normalized luciferase activity represented in the graphs was the result of dividing the relative light unit (RLU) values obtained for each inflammation-responsive vector by the mean RLU value obtained for the constitutive SFFV promoter vector under the same conditions.
6
ROS Generation in Neutrophils
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The generation of ROS in the neutrophils was measured using luminol-enhanced chemiluminescence (15 (link)). In brief, 3×105 neutrophils were suspended in 1 mL of HBSS. The cells were incubated for 30 minutes with saline, Ang-(1–7) (0.1 µmol/L), and Ang-(1–7) (0.1 µmol/L) combined with A779 (10 µmol/L), respectively, and then stimulated by 10 µmol/L of platelet-activating factor (PAF). ROS production was determined as the light released from the lumiphor luminol by a chemiluminometer (Autolumat LB953; Berthold Technologies, Bad Wildbad, Germany), and expressed as counts per second.
7
Luciferase Activity Quantification
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Cells were collected in PBS and lysed in luciferase assay buffer (25 mM Tris–phosphate pH 7.8, 8 mM MgCl2, 1 mM DTT, 1% Triton X-100 and 7% glycerol) during 15 min at room temperature in a horizontal shaker. Luciferase assay was performed using Luciferase Assay Reagent (Promega) according to the manufacturer’s instructions. Luciferase activity was measured using an Autolumat LB 953 (Berthold Technologies GmbH, Bad Wildbad, Germany) and normalised with protein concentration.
8
Superoxide Anion Detection in VSMCs
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The superoxide anion generated by NAD(P)H oxidase activity was determined using a chemiluminescence assay using 5 µM lucigenin and 100 µM NAD(P)H. VSMCs treated as described for the ROS detection experiments were homogenized in a lysis buffer (50 mM KH2PO4, 1 mM ethylene glycol tetraacetic acid, 150 mM sucrose, pH = 7.4). The reaction was initiated with the addition of a mixture of lucigenin and NAD(P)H to the protein sample in a final volume of 300 µl. Chemiluminescence was determined every 2.4 s for 5 min in a plate luminometer (Auto-Lumat LB 953, Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany). A buffer blank was subtracted from each reading, and the value of the area under the curve was used to quantify chemiluminescence. The data obtained (counts/s) were expressed as fold increases over the control situation.
9
Assessing NADPH Oxidase Activity in HAEC
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Lucigenin-enhanced chemiluminescence assay was used to determine NADPH oxidase activity in HAEC as we previously described.24 (link) Cells were stimulated with total microparticles (106 MPs/mL) isolated from patients pre-VEGFi and post-VEGFi treatment for different time periods. In some experiments, 30 min prior to stimulation with MPs, cells were pre-incubated with BQ123 and BQ788. In brief, stimulated cells were washed with ice-cold PBS and harvested in lysis buffer (20 mmol/L of KH2PO4, 1 mmol/L of EGTA, 1 μg/mL of aprotinin, 1 μg/mL of leupeptin, 1 μg/mL of pepstatin, and 1 mmol/L of PMSF). The sample of 50 µL were added to a suspension containing 175 μl of assay buffer (50 mmol/L of KH2PO4, 1 mmol/L of EGTA, and 150 mmol/L of sucrose) and lucigenin (5 μmol/L). Luminescence was measured with a luminometer (AutoLumat LB 953, Berthold) before and after stimulation with NADPH (100 µmol/L). A buffer blank was subtracted from each reading. The results are expressed as a fold change in arbitrary units per milligram of protein, as measured by the BCA assay.
10
NADPH Oxidase-Mediated O2•− Quantification
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