Stable cell lines transduced with Prx3, Trx2 and PGC-1α containing lentiviral vector or empty pRRL vector were plated in 96-well plates (Greiner Bio-One, Frickenhausen, Germany) and grown until confluent. Cells were treated with 200 μM of tbH2O2 for 6 hours. Hereafter, cell viability was assessed using the LIVE/DEAD Viability/Cytotoxicity kit (Invitrogen) according to the manufacturer’s protocol. Fluorescent signals were measured with the Fluostar Galaxy (BMG Labtech, Ortenberg, Germany) fluometer and the ratio between dead and live cells was calculated. Endogenous ROS production was assessed using 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein di-acetate, acetyl ester (CM-H2DCFDA; Invitrogen), a probe which turns fluorescent upon oxidation. Total fluorescence was measured with the Fluostar Galaxy (BMG Labtech, Ortenberg, Germany) and corrected for live cell number.
Fluostar galaxy
Manufactured by BMG Labtech
Sourced in Germany, United States
The FLUOstar Galaxy is a multi-mode microplate reader designed for sensitive fluorescence detection. It offers a broad range of excitation and emission wavelengths to accommodate a variety of fluorescent probes and assays.
Automatically generated - may contain errors
Lab products found in correlation
Fluostar optima, by BMG Labtech (7 mentions)
Fluostar omega, by BMG Labtech (4 mentions)
96 well plate, by Greiner (3 mentions)
Celltiter 96 aqueous non radioactive cell proliferation assay, by Promega (3 mentions)
Cytotox one homogeneous membrane integrity assay, by Promega (2 mentions)
Anti ha antibody, by Fortrea (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (2 mentions)
Mir control, by Thermo Fisher Scientific (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Cyquant cell proliferation assay, by Thermo Fisher Scientific (1 mentions)
96 well plate, by Corning (1 mentions)
384 well plate, by Corning (1 mentions)
Hq535 30m, by Chroma Technology (1 mentions)
Hq500 20x, by Chroma Technology (1 mentions)
Live dead viability cytotoxicity kit, by Thermo Fisher Scientific (1 mentions)
Glass 3881, by Corning (1 mentions)
Citrate synthase from porcine heart, by Merck Group (1 mentions)
Superdex 75 10 300 gl column, by GE Healthcare (1 mentions)
Z vad fmk, by Abcam (1 mentions)
84 protocols using fluostar galaxy
1
Assessing Oxidative Stress Resistance
2
Cytotoxicity Assay of Inflammatory Stimuli
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TR146 cells were seeded (2 × 104 cells/100 µL) in 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) and cultured for 24 h. Subsequently, the medium was replaced by IL-1α, IL-1β, LPS, and TNF-α diluted with serum-free DMEM and incubated for 24 h (n = 6). For each stimulant, different concentrations were used: (i) IL-1α (100–800 ng/mL), (ii) IL-1β (100–800 ng/mL), (iii) LPS (300–70,000 ng/mL), and (iv) TNF-α (100–800 ng/mL). To determine the cell viability, a CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega Corporation, Madison, WI, USA) was used according to the manufacturer’s instructions. After an incubation time of 4 h, the absorbance was measured at 490 nm using a UV-/VIS-plate reader (Fluostar Galaxy, BMG Labtech GmbH, Ortenberg, Germany). Untreated, blank-corrected wells represented 100% viability. The membrane integrity of TR146 cells was investigated by determining the lactate dehydrogenase (LDH) release using a CytoTox-ONE Homogeneous Membrane Integrity Assay (Promega) according to the manufacturer’s instructions. The fluorescence signal was measured at an excitation wavelength of 560 nm and an emission wavelength of 590 nm with a UV-VIS-plate reader (Fluostar Galaxy, BMG Labtech). Control wells representing 100% LDH release were treated with 2 µL of a lysis solution and all data were blank corrected.
3
Enzymatic Assays in Gingival Crevicular Fluid
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The 5 enzyme levels were assayed using fluorescent and chromogenic substrates. Each reaction was measured in a microplate reader (FLUOstar Galaxy; BMG Labtech GmbH, Offenburg, Germany). All chemicals in this study were from Sigma-Aldrich Company Ltd, Dorset, UK unless otherwise stated.
Standard curves for each enzyme were produced using serial dilutions of the following enzyme solutions in PBS pH 7.3: 100ng/ l Pro-MMP8 (Enzo Life Sciences Inc. Lausen, Switzerland activated with 1 l of 20 mM 4-amino-phenyl-mercuricacetate for 5 minutes at room temperature), 20 ng/ l neutrophil elastase, 16.6 ng/ l leukocyte cathepsin G, 100ng/ l pancreatic trypsin (containing 5mM di-thiothreitol) and 51ng/ l Clostridial sialidase.
For the above standard enzymes and the diluted clinical samples (10 l GCF), substrate was
Standard curves for each enzyme were produced using serial dilutions of the following enzyme solutions in PBS pH 7.3: 100ng/ l Pro-MMP8 (Enzo Life Sciences Inc. Lausen, Switzerland activated with 1 l of 20 mM 4-amino-phenyl-mercuricacetate for 5 minutes at room temperature), 20 ng/ l neutrophil elastase, 16.6 ng/ l leukocyte cathepsin G, 100ng/ l pancreatic trypsin (containing 5mM di-thiothreitol) and 51ng/ l Clostridial sialidase.
For the above standard enzymes and the diluted clinical samples (10 l GCF), substrate was
4
Kinetics of Amyloid-β Fibril Formation
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Fibril formation kinetics was studied by recording the ThT fluorescence over time in a plate reader (FLUOStar Galaxy from BMG Labtech, Offenberg, Germany). The fluorescence was measured using bottom optics in half-area 96-well polyethylene glycol-coated black polystyrene plates with clear bottom (Corning Glass, 3881) using a 440 nm excitation filter and a 490 nm emission filter. 3 μM of Aβ42 solution, with or without 0.6 or 1.5 μM of Bri2 or Bri3 BRICHOS protein, was supplemented with 10 μM ThT, 80 μl was added to each well, and the plate was sealed and immediately placed in the fluorescence reader at room temperature (RT), and incubated under quiescent conditions with readings made every 5 min.
5
Thioflavin T Fluorescence Assay
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Assays were initiated by placing the 96-well plate at 37°C under quiescent conditions in a plate reader (Fluostar Omega, Fluostar Optima, or Fluostar Galaxy; BMG Labtech). The ThT fluorescence was measured through the bottom of the plate with a 440-nm excitation filter and a 480-nm emission filter. The ThT fluorescence was followed for three repeats of each sample.
6
Stress-Induced Corticosterone Levels
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5-HT is known to influence the hypothalamus-pituitary-adrenal axis. Thus, plasma corticosterone levels were measured in non-stressed (immediately killed after removal from home cage) and stressed (killed 5 min after FST paradigm) animals (Steiner et al., 2008 (link)). Mice were decapitated between 3 pm and 5 pm, and the blood was collected in EDTA di-calcium containing collection tubes (KABE Labortechnik, Nümbrecht). Plasma was separated from blood cells by centrifugation at 13,000 rpm for 5 min at 4°C and was stored until use at −20°C. Corticosterone concentration was analyzed using the Corticosterone EIA Kit (IBL, Hamburg) and measured by the FLUOstar Galaxy from BMG Labtech.
7
EGF Aggregation Kinetics with Bri2 BRICHOS
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The WT N3 EGF1–5 and R133C mutants were diluted with 1 × PBS to 10 µM and then equilibrated at 37°C with and without recombinant Bri2 BRICHOS in 1:1 ratio. The aggregation kinetics was measured by reading the apparent increase in absorbance at 360 nm using a microplate reader (FLUOStar Galaxy from BMG Labtech, Offenberg, Germany) with shaking (240 s 300 rpm before each cycle; the interval for each cycle was 5 min).
8
Assessing Cell Proliferation and Drug Screening
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Cell proliferation was assessed by CyQUANT™ Cell Proliferation Assay (Thermofisher Scientific). Wide‐spectra drug screening was performed on 384‐well plates (Corning, NY, USA). The cells were seeded in the density of 20 000 cells·cm−2 and cultivated for 24 h. Next, the treatment by the range of concentrations of all drugs was performed with EpMotion® 5075 Automated Liquid Handling System (Eppendorf, Hamburg, Germany), and cells were cultivated for the next 48 h. CyQUANT™ Cell Proliferation Assay was performed in the endpoint to analyze cell proliferation. For the combined treatment analysis, the cells were seeded in the density of 20 000 cells·cm−2 into 96‐well plates (Corning). Twenty‐four hours later, the cells were treated with GEM (in MQ water) concentration range for 24 h. The next day, the CHK1 inhibitors SCH900776 (4 µm in DMSO) or MU380 (4 µm in DMSO) were added for 2 h, followed by complete media exchange. CyQuant assay was performed 48 h post‐treatments as recommended by the manufacturer. The fluorescence was detected at 520 nm on a plate reader Fluostar Galaxy (BMG Labtech, Ortenberg, Germany).
9
Salivary Protein and Mucin Quantification
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UWS and UWSRT without prior dialysis were used to determine the total protein concentration. For the determination of the mucin concentration in UWS and UWSRT respectively, dialysis was performed using a cellulose acetate membrane (MW cut-off of 12–14 kDa, Carl Roth, Germany) in 2000 mL 50mM NaCl for 12 h to separate the high molecular weight proteins [56 (link)].
Salivary whole protein and mucin concentration were determined using a standard Pierce BCA Protein Assay Kit (Thermo Scientific™ Pierce™, Waltham, MA, USA). This assay uses the state-of-the-art method of reducing Cu2+ to Cu+1 when in contact with proteins in an alkaline medium. The amount of Cu+1 can then be assessed via color reaction upon bicinchoninic acid addition at 562 nm. Diluted Albumin served as a standard reagent and Milli-Q®-water as blank. The BCA assay was carried out according to the standard protocol. Briefly, 25 µL of both UWS and UWSRT (each n = 6) were mixed with 200 µL working reagent in a 96-well-plate. The plate was shaken for 30 s on a plate shaker and incubated for 30 min at 37 °C under light exclusion. Absorbance was measured using a UV-/VIS plate reader (Fluostar Galaxy, BMG Labtech, Ortenberg, Germany) at 562 nm.
Salivary whole protein and mucin concentration were determined using a standard Pierce BCA Protein Assay Kit (Thermo Scientific™ Pierce™, Waltham, MA, USA). This assay uses the state-of-the-art method of reducing Cu2+ to Cu+1 when in contact with proteins in an alkaline medium. The amount of Cu+1 can then be assessed via color reaction upon bicinchoninic acid addition at 562 nm. Diluted Albumin served as a standard reagent and Milli-Q®-water as blank. The BCA assay was carried out according to the standard protocol. Briefly, 25 µL of both UWS and UWSRT (each n = 6) were mixed with 200 µL working reagent in a 96-well-plate. The plate was shaken for 30 s on a plate shaker and incubated for 30 min at 37 °C under light exclusion. Absorbance was measured using a UV-/VIS plate reader (Fluostar Galaxy, BMG Labtech, Ortenberg, Germany) at 562 nm.
10
CFTR Activity Measurement by YFP Assay
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CFTR activity was determined by the YFP microfluorimetric assay (details can be found in previous studies [12 (link),31 (link)]. Briefly, prior to the assay, cells were washed with PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, 1 mM CaCl2, and 0.5 mM MgCl2) and then incubated for 25 min with 60 µL of PBS plus forskolin (20 µM) and IVA (1 µM) at 37 °C, to maximally stimulate the CFTR channel. Cells were then transferred to a microplate reader (FluoStar Galaxy or Fluostar Optima; BMG Labtech, Offenburg, Germany), equipped with high-quality excitation (HQ500/20X: 500 ± 10 nm) and emission (HQ535/30M: 535 ± 15 nm) filters for YFP (Chroma Technology, Bellows Falls, VT, USA). Each assay consisted of a continuous 14-s YFP fluorescence recording with 2 s before and 12 s after injection of 165 µL of an iodide-containing solution (PBS with Cl− replaced by I−; final I− concentration 100 mM). Data were normalized to the initial background-subtracted fluorescence. To determine the I− influx rate, the final 11 s of the data for each well were fitted with an exponential function to extrapolate the initial slope (dF/dt).
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