As a marker of leukocyte activity, we evaluated the concentration of myeloperoxidase (MPO) in the BAL 3 and 24 h after LPS administration. Additionally, MPO release from isolated murine neutrophils from bone marrow and human neutrophils from healthy volunteers was determined. MPO was measured colorimetrically at 405 nm by a plate reader (Tecan Reader Infinite M200PRO). Reactive oxygen species (ROS) were detected by dihydroethidium (D7008; Sigma-Aldrich) with a flow cytometry-based method under the indicated conditions (19 (link)).
Reader infinite m200pro
Manufactured by Tecan
Sourced in Switzerland
The Reader Infinite M200PRO is a microplate reader designed for versatile and precise absorbance measurements. It features a wide wavelength range, advanced optics, and intuitive software to facilitate a variety of applications in life science research and diagnostics laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Phorbol 12 myristate 13 acetate pma, by Merck Group (2 mentions)
Luminol, by Thermo Fisher Scientific (2 mentions)
I control 1, by Tecan (2 mentions)
Nunc f96 microwell polystyrol plate, by Thermo Fisher Scientific (2 mentions)
D7008, by Merck Group (1 mentions)
Dy4517, by R&D Systems (1 mentions)
Eia kit, by Cayman Chemical (1 mentions)
Zetasizer nano z, by Malvern Panalytical (1 mentions)
8 protocols using reader infinite m200pro
1
Neutrophil Activation Markers in LPS-Induced Lung Injury
2
Quantification of Neutrophil Extracellular Traps
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To quantify the NET formation, we performed various complementary assays. We used the Quanti-iT double-stranded (ds) DNA PicoGreen assay kit (P7589; Invitrogen; Waltham; MA, USA) and murine neutrophil elastase (NE) (DY4517; R&D Systems; USA) ELISA to determine the release of extracellular dsDNA NE in the peritoneal lavage. Further, we evaluated the release of dsDNA (Quanti-iT dsDNA PicoGreen; P7589; Invitrogen; Waltham; USA) and human NE (DY9167; R&D Systems; Minneapolis; USA) in our in vitro experiments with human PMNs.
We determined the release of myeloperoxidase (MPO) as an activation marker for PMNs in the peritoneal lavage of mice four hours after zymosan and fecal administration. The MPO release from human PMNs from healthy volunteers was also determined at indicated conditions. MPO was measured colorimetrically at 405 nm by a plate reader (Tecan Reader Infinite M200PRO; Männedorf; Switzerland).
We determined the release of myeloperoxidase (MPO) as an activation marker for PMNs in the peritoneal lavage of mice four hours after zymosan and fecal administration. The MPO release from human PMNs from healthy volunteers was also determined at indicated conditions. MPO was measured colorimetrically at 405 nm by a plate reader (Tecan Reader Infinite M200PRO; Männedorf; Switzerland).
3
Colorimetric ATPase Assay for ABC Transporters
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A colorimetric ATPase assay was carried out to test the effect of PT on ABC transporters. Membranes with human BCRP were purchased from Corning Life Sciences (NY, United States). The assay was conducted following the manufacturer’s protocol. We previously described the ATPase protocol in details (Ooko et al., 2016 (link); Hamdoun et al., 2017 (link)). Briefly, a reaction mixture composed of membranes, PT concentration, MgATP and assay buffer was incubated for 20 min at 37°C. To stop the reaction, 10% SDS was added. Afterward, a color reagent was added to the wells, in order to measure inorganic phosphate using Tecan Reader Infinite m200 Pro. The assay was performed in triplicate. Nunc transparent flat-bottomed plates were used for the measurements. Sulfasalazine was used as positive control.
4
Quantifying COX-2 Inhibitory Activity
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An enzyme immunoassay (EIA) kit (#560101, Cayman Chemicals) was performed to inhibit human recombinant COX-2. Assays were performed according to the manufacturer's protocol. Asa, asplatin and OxAsaOAc were used with a final concentration of 250 μM. Celecoxib was used with a final concentration of 10 μM.
Absorbance was measured at 410 nm with a Tecan Reader infinite® M200Pro (Tecan Group Ltd, Switzerland). COX-2 inhibition was calculated as instructed by the manufacturer protocol.
Absorbance was measured at 410 nm with a Tecan Reader infinite® M200Pro (Tecan Group Ltd, Switzerland). COX-2 inhibition was calculated as instructed by the manufacturer protocol.
5
MTT Assay for Compound Cytotoxicity
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Cells were seeded in 96-well microtiter plates and allowed to adhere overnight. Subsequently, cells were exposed in triplicates to the compounds (pre-dissolved in DMSO, final DMSO concentration <0.5%) in the indicated concentrations. After 72 h cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (EZ4U, Biomedia, Vienna, Austria) according to the manufacturer's recommendation. Absorbance was measured at 450 nm (620 nm as a reference) with a Tecan Reader infinite® M200Pro (Tecan Group Ltd, Switzerland). Data were analyzed using Graph Pad prism (version 8.0.1) to calculate IC50 values, as a parameter for cytotoxicity resulting in 50% reduction of cell viability compared to the untreated control cells.
6
Luminol-based Quantification of Neutrophil-derived ROS
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The sum of intra- and extracellular MPO-derived ROS was measured by using the luminol-amplified chemiluminescence assay. Neutrophils (400,000 cells/sample) were resuspended in complete medium without FCS and seeded in a flat-bottom white 96-well plate (NuncTM F96 MicroWellTM polystyrol plate, Thermo Fisher). Subsequently, 60 μM luminol (Invitrogen, Germany) was added, and the cells were stimulated by the addition of 20 nM phorbol 12-myristate 13-acetate (PMA; Sigma Aldrich). The chemiluminescence resulting from ROS release was analyzed immediately by an Infinite M200pro-Tecan reader (Tecan, Männedorf, Switzerland) and Tecan i-control 1.7 software. ROS release was monitored every minute for a period of 1 h at 37°C and 5% CO2.
7
Quantifying Neutrophil-Derived ROS Release
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The sum of intra- and extracellular MPO-derived ROS (28 ) was measured by using a luminol-amplified chemiluminescence assay. Polarized neutrophils (400,000 cells/sample) were resuspended in complete medium without FCS and seeded in a flat-bottom white 96-well plate (NuncTM F96 MicroWellTM polystyrol plate, Thermo Fisher). Subsequently, 60 μM luminol (Invitrogen, Germany) was added, and the cells were stimulated by the addition of 20 nM phorbol 12-myristate 13-acetate (PMA; Sigma Aldrich). The chemiluminescence resulting from ROS release was analyzed immediately by an Infinite M200pro-Tecan reader (Tecan, Männedorf, Switzerland) and Tecan i-control 1.7 software. ROS release was monitored every minute for a period of 1 h at 37°C and 5% CO2. Extracellular superoxide was detected by using a lucigenin-amplified chemiluminescence assay (28 ). This assay was performed the same way as the luminol assay, but with 0.2 mM lucigenin (Alexis, Loerrach, Germany) instead of luminol.
8
Particle Characterization of Nanomaterials
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Particle size and size distribution, zeta potential, average hydrodynamic diameter (Dh), and polydispersity index of the particles were measured by dynamic light scattering using a Zetasizer Nano-ZS instrument (Malvern Instruments, Malvern, UK) at 25°C. The machine was equipped with a 633 nm He–Ne laser and a detector at an angle of 173°. The samples were diluted and sonicated in a water bath prior to size analysis. Iron concentration was measured using 1,10-phenanthroline assay as described previously.34 Finally, the absorbance was detected at 510 nm using an Infinite M200 Pro TECAN reader (TECAN, Germany).
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