The ABTS stock solution was prepared by adding 7 mM of ABTS with 8.75 mM of potassium persulfate and incubated in dark at room temperature for 16 h. Before use, the ABTS stock solution was diluted to 104.14 µM ABTS solution. Then, 20 µL of RPE or standard solution were mixed with 280 µL of 104.14 µM ABTS solution. After being mixed, the plate was incubated in dark at RT for 6 min and the absorbance was measured at 734 nm with spectrophotometer (LUMIstar, BMGLABTECH, Ortenberg, Germany).
Lumistar
Manufactured by BMG Labtech
Sourced in Germany
The LUMIstar is a multi-mode microplate reader designed for luminescence detection. It provides accurate and sensitive measurements of bioluminescence, chemiluminescence, and other luminescent assays.
Automatically generated - may contain errors
Lab products found in correlation
Caspase glo 3 7 assay, by Promega (3 mentions)
Bactiter glo microbial cell viability assay, by Promega (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Nad nadh glo assay, by Promega (1 mentions)
Culturplate, by PerkinElmer (1 mentions)
Jetpei, by Polyplus Transfection (1 mentions)
Hiperfect transfection reagent, by Qiagen (1 mentions)
White 96 well plate, by Thermo Fisher Scientific (1 mentions)
Nanoluc substrate furimazine, by Promega (1 mentions)
Clariostar multilabel plate reader, by BMG Labtech (1 mentions)
Celltiter glo luminescent cell viability assay, by Promega (1 mentions)
24 well plate, by Sarstedt (1 mentions)
14 protocols using lumistar
1
ABTS-Based Antioxidant Assay
2
NAD+ and NADH Quantification Assay
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5x105 cells were primed and stimulated as indicated. Subsequently, cells were pelleted and resuspended in 200µL cold PBS (Thermo Fisher Scientific Inc., Waltham, USA) and 200µL cold base solution (100mM sodium carbonate, 20mM sodium bicarbonate, 0.05% Triton X-100, 10mM nicotinamide, and 1% dodecyltrimethylammonium bromide [all from Merck KGaA, Darmstadt, Germany)] and stored at -80°C until further analysis. Directly before measurement, each 50µL sample were heated at 60°C for 15min either together with 25µL 0.4N HCl (Honeywell, Morristown, USA) or alone to destroy the NADH or NAD+ respectively. The acidified sample was neutralized with 25µL 0.5M Tris base (Carl Roth, Karlsruhe, Germany). To adjust volume and composition 25µL HCl and 25µL 0.5M Tris base were added to the other sample as well. NAD+ and NADH levels were assessed using the NAD/NADH-Glo Assay (Promega, Mannheim, Germany) as indicated by the manufacture. Measurement was performed on a LUMIstar® (BMG Labtech, Ortenberg, Germany) with a gain of 4095.
3
DPPH Radical Scavenging Assay
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For the DPPH assay, 20 µL of RPE was added into 280 µL of freshly prepared 107.14 µM DPPH solution. Trolox was used as a standard control. The mixture was incubated in dark at room temperature for 30 min and the absorbance was measured at 517 nm with spectrophotometer (LUMIstar, BMGLABTECH, Ortenberg, Germany).
4
Bacterial Viability Assay using BacTiter-Glo
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The bacterial cell viability was assessed with the BacTiter-Glo™ Microbial Cell Viability assay (Promega, Mannheim, Germany), according to the manufacturer’s protocol. Shortly, bacterial cells were grown O/N at 37 °C and 250 rpm and then diluted 1:100 in KPi buffer. Then, 150 µl from the KPi diluted cells were centrifuged in 1 ml tubes at 6200×g at 4 °C for 15 min. Subsequently, the supernatant was removed, and the pelleted cells were then resuspended with 150 µl of either KPi buffer (negative control) or 150 µl of JAMF1 IBs at 1, 3, 5 and 10 µM. After 5 h incubation at 37 °C in a 96-well plate, 100 µl were taken and mixed with 100 µl of the BacTiter-GloTM reagent. Finally, luminescence was measured in a microplate luminometer (LUMIstar®, BMG LABTECH. Ortenberg, Germany). The measured arbitrary luminescence values were normalized against the control (KPi treatment).
5
Neutrophil Oxidative Burst Assay
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Freshly isolated neutrophils from healthy donors were incubated in duplicate wells in a white 96-well plate at a density of 105 cells/well in HBSS with Ca2+/Mg2+ + 0.1% Glucose + 1% human serum albumin with 100 μΜ lucigenin at 37 °C and stimulated with 1 ng/ml TNF-α for 30 min followed by 1 μΜ fMLF. Lucigenin enhanced chemiluminescence was measured in a microplate reader (Lumistar, BMG Labtech) every 30 s for 60 min and measurements expressed as relative light units (RLUs). For HA, G6PDi, methylglyoxal, pyruvate, and lactate treatments, cells were pre-incubated with the reagents for 1 h.
6
Cardiac Oxidative Stress Measurement in Mice
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Cardiac LV tissues of C57BL/6J mice in each group (NCD WT, NCD Nox2KO, HFD WT and HFD Nox2KO; n = 6 per group) were isolated. Disintegrated and homogenised LV tissue was analysed for O2 •- generation by means of lucigenin-chemiluminescence (Lumistar, BMG Labtech GmbH, Ortenberg, Germany) using 5 μM of lucigenin as previously described [15] (link). A O2 •-scavenger, Tiron (1 M), confirmed O2 •-generation.
Prior to measurement of O2 •-, the inhibitors targeting nitric oxide synthases (L-NAME, 100 μM), the mitochondrial complex-1 enzymes (Rotenone, 50 μM), xanthine oxidases (Oxypurinol, 250 μM) and flavoproteins (DPI, 20 μM or ds-tat, 10 μM) were administered. In situ production of O2 •-in intact LV cardiac cells was measured using the redox indicator DHE. Intact LV sections (6 µm) were produced using a cryostat (Bright Instruments, Bedfordshire, UK) from tissue (3 x 3 mm) obtained from a lower LV region of the harvested mouse hearts. DHE fluorescence was performed as previously described [16] (link). Within 7 min of staining, images were captured using the Oxion inverted fluorescent microscope (Euromex, Arnhem, Netherlands) at excitation 518 nm, emission 605 nm. Using HCImageLive (x64) software, fluorescence intensity was quantified, calculated from at least 10 random fields (254.3 × 254.3 μm) for each section, 3 sections per sample and 6 animals per group.
Prior to measurement of O2 •-, the inhibitors targeting nitric oxide synthases (L-NAME, 100 μM), the mitochondrial complex-1 enzymes (Rotenone, 50 μM), xanthine oxidases (Oxypurinol, 250 μM) and flavoproteins (DPI, 20 μM or ds-tat, 10 μM) were administered. In situ production of O2 •-in intact LV cardiac cells was measured using the redox indicator DHE. Intact LV sections (6 µm) were produced using a cryostat (Bright Instruments, Bedfordshire, UK) from tissue (3 x 3 mm) obtained from a lower LV region of the harvested mouse hearts. DHE fluorescence was performed as previously described [16] (link). Within 7 min of staining, images were captured using the Oxion inverted fluorescent microscope (Euromex, Arnhem, Netherlands) at excitation 518 nm, emission 605 nm. Using HCImageLive (x64) software, fluorescence intensity was quantified, calculated from at least 10 random fields (254.3 × 254.3 μm) for each section, 3 sections per sample and 6 animals per group.
7
Bacterial Cell Viability Assay
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Bacterial cell viability was determined with a BacTiter-Glo™ Microbial Cell Viability assay (Promega). Briefly, bacterial cells were grown O/N at 37 °C and 250 rpm and then diluted 1:100 in 10 mM KPi buffer. After that, 150 μL from the KPi diluted cells were centrifuged in 1 ml tubes at 6200× g at 4 °C for 15 min. The supernatant was removed, and the pelleted cells were resuspended with 150 μL of either KPi buffer (negative control) or 150 μL of 1 and 3 μM of the synthetic HD5 peptide or the protein constructs (HD5-GFP, JAMF1, JAMF1.2 or JAMF2). After 0.5 h, 2 h or 5 h incubation at 37 °C in a 96-well plate, 100 μL were taken and mixed with 100 μL of the BacTiter-Glo™ reagent for 5 min. Luminescence was measured in a microplate luminometer (LUMIstar®, BMG LABTECH). The measured luminescence values (arbitrary units) were normalized to the control (KPi buffer treatment; equivalent to 100% bacterial survival). HD5 (PeptaNova) served as a control.
8
BRET Assay for CGRP Receptor Activation
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HEK293T cells stably expressing the CAMYEL BRET sensor (~2 × 106) were seeded into 90 mm Petri dish (Corning™, USA) in DMEM/FBS/Geneticin and incubated in 5% CO2 and 95% O2 (24 h, 37 °C). Prior to the transfection, the medium was changed to fresh DMEM/FBS/Geneticin and rat CLR/RAMP1 was transfected (2.5 µg CLR/RAMP1 DNA/dish) using JetPEI (Polyplus Transfection, France) at a 1:6 ratio. After 24 h, cells were plated in poly-L-lysine coated black 96 well CulturPlate (Perkin Elmer, USA) and incubated in 5% CO2 and 95% O2 (24 h, 37 °C). BRET was assessed using a LUMIstar (BMG LABTECH, Germany). On the day of the assay, cells were equilibrated in HBSS for 30 min, supplemented with 12 mM HEPES at 37 °C in CO2-free incubator. DIPMA-MK-3207 or free MK-3207 was incubated for 25 min, followed by the addition of coelentrazine-h (50 µM) for 5 min. Baseline was then measured for 5 min, followed by stimulation with CGRP (100 nM, ~EC50), vehicle (HBSS) or forskolin (1 µM, positive control), and further measurements for 5 min. Buffer was then replaced by HBSS with coelentrazine-h (50 µM) and measurements were resumed for further 25 min.
9
Caspase 3/7 Activity Assay in Breast Cancer
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Caspase-Glo 3/7 assay (Promega, Madison, WI, USA) was applied to measure the activity of caspase 3 and 7 according to the manufacturer’s instructions. For transient transfection experiments, BC cell lines were seeded in 96-well plates and transfected using the reverse transfection protocol and HiPerFect Transfection Reagent (Qiagen) in five technical replicates. Caspase 3 and 7 activity was measured 48 h after transient transfection. Lentiviral stable-transduced cells were also seeded in 96-well plates, and apoptosis was measured after 48 h. After adding the substrate, luminescence was measured using a luminometer (LumiStar, BMG Labtech, Ortenberg, Germany).
10
Caspase-3/7 Activity Quantification
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Activity of caspases 3 and 7 was determined by Caspase-Glo 3/7 assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. All three ccRCC cell lines were seeded in 96-well plates and transfected with the two different siRNAs against PANTR1 and the controls as described above. Luminescence was recorded using white 96-well plates (Thermo Scientific) and a luminometer (LumiStar, BMG Labtech, Ortenberg, Germany) 72 and 96 h after transfection. All experiments were performed in four independent repeats. Differences in luminescence between the control condition and the two corresponding transfected cell lines were calculated using the ANOVA method.
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