The metabolic activity of Ba/F3 and MOLM13 cells was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTS, Promega, Madison, WI, USA). In detail, 20 µL MTS reagent was added to the cell suspension in 96-well plates. After centrifugation and incubation for 1 to 4 h, readouts were performed with the Clariostar plate-reader and analyzed by Mars Data Analysis Software (BMG Labtech, Ortenberg, Germany). As an additional tool for comparing cell viability, PrestoBlue reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used. After incubation of the 90 µl of cell suspension in 96-well plates, 10 µL of PrestoBlue reagent were added and analysis was performed after 30 min incubation by the Clariostar plate-reader. The data were processed Mars Data Analysis Software (BMG Labtech).
Mars data analysis software
Manufactured by BMG Labtech
Sourced in Germany, United Kingdom, France, United States
MARS Data Analysis Software is a tool developed by BMG Labtech for the analysis of data obtained from their line of laboratory equipment. The software provides essential functionalities for managing, processing, and visualizing experimental data.
Automatically generated - may contain errors
Lab products found in correlation
Fluostar omega microplate reader, by BMG Labtech (16 mentions)
Fluostar omega, by BMG Labtech (9 mentions)
Clariostar microplate reader, by BMG Labtech (9 mentions)
96 well black walled microplate, by Corning (6 mentions)
Clariostar, by BMG Labtech (5 mentions)
Fluostar optima, by BMG Labtech (4 mentions)
Spectrostar nano, by BMG Labtech (4 mentions)
Celltiter glo luminescent cell viability assay, by Promega (3 mentions)
Clariostar plus plate reader, by BMG Labtech (2 mentions)
384 well plate, by Greiner (2 mentions)
Fluostar omega plate reader, by BMG Labtech (2 mentions)
Clariostar plate reader, by BMG Labtech (2 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (2 mentions)
Pherastar fs plate reader, by BMG Labtech (2 mentions)
Thiazolyl blue tetrazolium blue mtt, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by AppliChem (2 mentions)
Fluxor, by Thermo Fisher Scientific (2 mentions)
Fluxor, by BMG Labtech (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
89 protocols using mars data analysis software
1
Cell Viability Assays for Ba/F3 and MOLM13 Cells
2
Fluorescence-Based Quantification of LAMP
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The fluorescence labeling with TAMRA-dUTP was analyzed with the MARS data analysis software (BMG LABTECH) and FLUOstar Omega microplate reader (BMG LABTECH). Using the fluorescence spectrometer, fluorescence was measured with 15 µl of purified LAMP products and using the following parameters: 544 nm excitation and 590 nm emission filter; gain = 1500. All measurements were conducted as triplicates.
A standard calibration line for the incorporation of TAMRA was created by measuring free TAMRA-dUTP at a dilution range of 10 µM to 4.88 nM.
The evaluation of SYBR Green intercalation was also conducted to perform a relative quantification of amplicons produced within a LAMP reaction. To carry out this analysis, the MARS data analysis software (BMG LABTECH) and the FLUOstar Omega microplate reader (BMG LABTECH) were used. The fluorescence spectrometer was employed to analyze 15 µl of purified LAMP products, using the following settings: an excitation wavelength of 485 nm and an emission wavelength of 520 nm with a gain value of 1000. All measurements were conducted as triplicates.
A baseline signal of 1 × Isothermal buffer (New England Biolabs, B0537) was included in all fluorescence measurements to help detect any intrinsic fluorescence or background noise.
A standard calibration line for the incorporation of TAMRA was created by measuring free TAMRA-dUTP at a dilution range of 10 µM to 4.88 nM.
The evaluation of SYBR Green intercalation was also conducted to perform a relative quantification of amplicons produced within a LAMP reaction. To carry out this analysis, the MARS data analysis software (BMG LABTECH) and the FLUOstar Omega microplate reader (BMG LABTECH) were used. The fluorescence spectrometer was employed to analyze 15 µl of purified LAMP products, using the following settings: an excitation wavelength of 485 nm and an emission wavelength of 520 nm with a gain value of 1000. All measurements were conducted as triplicates.
A baseline signal of 1 × Isothermal buffer (New England Biolabs, B0537) was included in all fluorescence measurements to help detect any intrinsic fluorescence or background noise.
3
Macrophage Stimulation by S. aureus
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For macrophage stimulation, 7.5 × 104 cells/well of J774.A1 murine macrophages (45 (link)) (a kind gift of Jos van Putten, Utrecht University) were seeded in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FCS (Bodinco B.V., Alkmaar, the Netherlands) at 37°C, 5.0% CO2. After overnight adherence, cells were stimulated with live or heat-killed (1 h at 80°C) S. aureus (multiplicity of infection [MOI] of 20:1) in the presence or absence of CATH-2 (0.01 to 2.5 µM) for 2 h at 37°C in DMEM plus 10% FCS, after which supernatants were collected to determine TNF-α levels by enzyme-linked immunosorbent assay (ELISA; R&D Systems Minneapolis, MN). TNF-α ELISAs were performed according to the manufacturer’s protocol. Samples were measured with a FLUOstar omega microplate reader (BMG Labtech GmbH, Ortenberg, Germany) and analyzed with MARS data analysis software (BMG Labtech GmbH). Measurements of optical density at 450 nm (OD450) were corrected by subtracting OD570 measurements. In addition to macrophage stimulation, mixtures of S. aureus and CATH-2 in DMEM plus 10% FCS were incubated for 2 h at 37°C, followed by serial dilution and spread plating on tryptone soy agar petri dishes to determine bacterial viability after overnight incubation.
4
Clonogenic Assay with AlamarBlue
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100 µL of 0.3% agar-agar in growth medium was added to a 96-well microculture plate (Sigma-Aldrich, TPP Zellkultur. Testplatte, Trazadingen, Switzerland) and left to solidify at room temperature for 30 min. Immediately after irradiation, cell suspensions were gently mixed with 0.2% agar-agar preheated on water at 43 °C. The final concentration of cells per well was 6 × 102 cells/100 µL. Immediately after solidification, 50 µL of 0.3% agar-agar in growth medium was added to each well as a feeding layer and allowed to solidify for 30 min at room temperature. Cells were incubated under standard CO2 incubator conditions (5% CO2, 37 C) for 7 days, after which 10% AlamarBlue reagent (Invitrogen, Frederick, MD, USA) was added to each well and cells were incubated for 2 h at 5% CO2 and 37 °C. The fluorescence of the AlamarBlue reagent was determined using a CLARIOstar instrument (BMG LABTECH, Ortenberg, Germany) at excitation/emission wavelengths of 530/590 nm. The obtained data were processed using the MARS data analysis software (BMG LABTECH, Ortenberg, Germany).
5
Mouse Cytokine ELISA Assay
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ELISA DuoSets for mouse TNF-α and mouse IL-6 were obtained from R&D Systems (Minneapolis, MN, USA). Samples were diluted in phosphate-buffered saline (PBS) with 1% bovine serum albumin (BSA), pH 7.4, before analysis. ELISAs were performed according to the manufacturer's protocol. For ELISA plate analysis, absorbance was determined at an OD at 450 nm (OD450) and was corrected at OD570. Absorbance was determined with a FLUOstar Omega microplate reader (BMG Labtech GmbH, Ortenberg, Germany) and analyzed with MARS data analysis software (BMG Labtech GmbH).
6
CFTR-mediated Iodide Influx Assay
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FRT cells expressing wild type- or ΔF508-CFTR with the halide sensor YFP-H148Q/I152L were plated in 96-well black-walled microplates (Corning Inc., Corning, NY) at a density of 2 x104cells per well. FRT-WT-CFTR-YFP cells were incubated for 48 h at 37°C, and FRT-ΔF508-CFTR-YFP cells were incubated for 24 h at 27°C after 24 h incubation at 37°C to rescue ΔF508-CFTR localization. Assays were done using FLUO star Omega microplate reader (BMG Labtech, Ortenberg, Germany) and MARS Data Analysis Software (BMG Labtech). Each well of a 96-well plate was washed 3 times in PBS (200 μL/wash). 100 μL PBS was added to each well. Forskolin (0.1 and 10 μM for WT- and ΔF508-CFTR, respectively) and test compounds (1 μL) were added to each well. After 10 min, 96-well plates were transferred to the microplate reader preheated to 37°C for fluorescence assay. Each well was assayed individually for CFTR-mediated I- influx by recording fluorescence continuously (200 ms per point) for 2 s (baseline), then 100 μL of 140 mM I- solution was added at 2 s and then YFP fluorescence was recorded for 14 s. Initial iodide influx rate was determined from the initial slope of fluorescence decrease, by nonlinear regression, following infusion of iodide.
7
Cell Polarization Measurement Protocol
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For the cell polarization measurements, 5 × 104 stably or transiently transfected GripTite HEK293 cells were seeded into black/clear 96-well plates (Falcon; Thermo Fischer Scientific) and cultured for 24 h in 100 µL FluoroBrite (Thermo Fisher Scientific), 2% FBS, 2 mM L-glutamine, and 50 units/mL Pen-Strep at 37 °C and 5% CO2. For in vitro polarization measurements, individual recombinant proteins, or mixtures thereof, at a final concentration of 10 µM, were diluted into 20 mM Tris⋅HCl (pH 7.5), 150 mM NaCl, and 10 mM dithiothreitol (DTT) in a final volume of 20 µL. The mixture was incubated for 10 min and measured in a black/clear 384-well plate (BD Falcon), using a PHERAstar FS microplate reader (BMG Labtech) equipped with a fluorescence polarization filter, excitation at 430 nm and emission at 480 nm. Polarization was calculated using MARS data analysis software (BMG Labtech) according to the following equation:
The G factor (compensation factor for the plate reader) was set automatically for each measurement based on gain adjustment settings.
The G factor (compensation factor for the plate reader) was set automatically for each measurement based on gain adjustment settings.
8
Arsenite-Induced Oxidative Stress in HaCaT Cells
9
Continuous Fluorometric Assay for TcTS
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The hydrolytic activity of TcTS was determined using a continuous fluorometric assay by measuring the rate of release of 4-methylumbelliferone from the substrate 2'-(4methylumbelliferyl)--D-N-acteylneuraminic acid (MuNANA) as described previously [19] . Fluorescence was measured on a FLUOstar Omega (BMG labtech) using excitation and emission filters at 360 and 460 nm, respectively. All assays were run in 20mM Tris-HCl and 50mM NaCl at pH 7.5, containing 0.01% Triton X100 at 35°C. TcTS (50 g/mL) was preincubated with each compound over at least seven inhibitor concentrations (ranging from 1 M to 15 mM) for 10 minutes at 35°C, then the assay initiated by the addition of MuNANA (200 M) to give a final reaction volume of 100 L. The fluorescence was analysed using MARs data analysis software (BMG labtech), where a linear region of the raw data curve was selected and the slope calculated (RFU/min) which represents hydrolytic activity of the enzyme. The IC50 values were determined as the concentration of inhibitor required to reduce hydrolytic activity of TcTS by 50% compared to the control (no inhibitor). All assays were repeated in three (n=3) independent experiments.
10
Lentiviral Particle Production and Titration
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Viral particles were produced by transfection of 293FT cells with 0.4 μg of CMV-VSVG DNA, 2.6 μg of pSIV3+, pNLAD8, or pJK7312As DNA, and 8 μL of TransIT-293 (Mirus Bio, CAT# MIR2700) per well in 6-well culture plates (Dutscher, CAT#9206). Medium was replaced after 24 h. Viral supernatants were harvested the next day, filtered at 0.45 μm, and either used fresh or concentrated via ultracentrifugation and then frozen at −80 °C. Viral titers were measured using GHOST X4R5 cells as described previously35 (link) or via HIV-1 p24 or SIV p27 ELISAs (XpressBio, CAT#XB-1010 or CAT#SK845, respectively) using a CLARIOstar Microplate Reader (BMG Labtech) with MARS Data Analysis Software (BMG Labtech). The pNLAD8 ELA20 Δenv and pJK7312As ELA20 Δenv viruses were purified and concentrated on a 20% sucrose cushion via ultracentrifugation at 4 °C. Pellets were resuspended in AIM V medium (Thermo Fisher Scientific, CAT#10297582) and then frozen at −80 °C.
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