Quantitative measurements of AChE enzymatic activity were made using a modified Ellman method (Ellman et al., 1961 (link); Rosenfeld et al., 2001 (link)). Stock solutions were acetylthiocholine iodide, used as the enzymatic substrate (ATH; 1.7 mg/ml in PBS, Sigma-Aldrich), 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB; 0.8 mg/ml in PBS, Sigma-Aldrich). Briefly, brains were rapidly dissected from either WT or J20 mice. Neocortex was isolated, weighed, and then homogenized using a Pellet Pestle (Sigma, Z 359971) in nine volumes of 0.1 M sodium phosphate buffer (pH 7.4; Patel et al., 2014 (link)). Five microliters of brain homogenate was aliquoted into each well of a 96-well plate, volume made up to 200 µl with PBS. DTNB (50 µl from stock) was added, followed by 50 µl of ATH substrate from stock. Measurement of absorption at 450 nm began immediately (<2 h from dissection) and was measured every 5 min for up to 30 min using a MRX microplate reader (Dynex Technologies). Thiocholine production in the test wells was expressed in units of nmol/min, calibrated with reference to the absorbance change over a range of concentrations giving a linear response using glutathione as the DTNB reactant (Eyer et al., 2003 (link)). Neostigmine (10 µM, Sigma-Aldrich) was used to completely inhibit AChE activity and establish that there was no baseline drift during the measurements.
Mrx microplate reader
Manufactured by Dynex
Sourced in United States, Germany, United Kingdom
The MRX microplate reader is a versatile instrument designed for the measurement of absorbance, fluorescence, and luminescence in microplate format. It features a high-performance optical system and supports a wide range of microplate sizes and detection modes to accommodate a variety of assays and applications.
Automatically generated - may contain errors
Lab products found in correlation
Methyl thiazolyl tetrazolium (mtt), by Merck Group (4 mentions)
O phenyldiamine, by Merck Group (2 mentions)
Immulon 4, by Dynex (2 mentions)
Mouse iaa elisa kit, by Biorbyt (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Pellet pestle, by Merck Group (1 mentions)
Neostigmine, by Merck Group (1 mentions)
5 5 dithiobis 2 nitrobenzoic acid dtnb, by Merck Group (1 mentions)
Human albumin, by Fortis Life Sciences (1 mentions)
Ammonium chloride, by Merck Group (1 mentions)
Dc protein assay system, by Bio-Rad (1 mentions)
Td 20 20 luminometer, by Turner Designs (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Horseradish peroxidase hrp conjugated anti goat igg antibody, by R&D Systems (1 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions)
Luminex assay, by R&D Systems (1 mentions)
Bio plex 200 system, by Bio-Rad (1 mentions)
Cck 8 kit, by Merck Group (1 mentions)
Cell counting kit 8 cck 8 kit, by Merck Group (1 mentions)
Mouse albumin elisa quantitation kit, by Fortis Life Sciences (1 mentions)
28 protocols using mrx microplate reader
1
Quantitative Measurement of AChE Activity
2
Evaluating Hepatocyte Response to AAT
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To determine if AAT (2–8 mg/ml) affected hepatocyte viability and function, MTT, albumin and urea assays were carried out. AAT was dissolved in 100 μl William's E (WE) medium and added to the cell culture media. Hepatocyte viability was determined using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Briefly, cells were cultured for 24 h at 37 °C, with 5% CO2, supernatant was removed and cells were cultured with serum-free medium containing 0.5 mg/ml of MTT (Sigma-Aldrich, Dorset, UK) for 4 h. After removal of the MTT, the produced formazan was dissolved in DMSO and the optical density read at 570 nm on a Dynex MRX microplate reader. For albumin quantification, cell culture medium was collected 12 h post-plating and enzyme immunoassays carried out for human albumin (Bethyl Laboratories, Inc., TX, USA). Ammonia metabolism was measured using a QuantiChrom™ Urea Assay kit (Universal Biologicals, Cambridge, UK). Cells were washed with PBS and incubated with 5 mM ammonium chloride (Sigma-Aldrich, Dorset, UK) for 6 h before measurement of urea synthesis.
3
Dual-Luciferase Assay for Gene Expression
4
MTT Assay for Evaluating Cell Viability
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The MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma, St. Louis, MO, USA) assay was utilized as a method to determine the cell vitality. NRK-52E cells with 1 × 105 cells/well were incubated with MV or DEX-MV for 1.5 h in the incubator with MTT. Then, the treated NRK-52E cells were exposed to hypoxia (5% CO2, 1% O2 and 94% N2) for 4 h, then to reoxygenation (5% CO2, 21% O2 and 74% N2) for 2 h. The optical density value was determined and read by an ELISA reader (MRX Microplate Reader, Dynex Technologies, Inc., Chantilly, VA, USA) at 570 nm.
5
Nitric Oxide Production Quantification
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To determine nitric oxide production, the level of nitrite, a stable oxidative end product of nitric oxide, was measured using a previously described colorimetric method [14 (link)] with minor modifications. The VSMCs (5 × 105 cells/dish) were seeded in DMEM containing 10% FBS in 6-cm culture dishes, and grown until the monolayer was confluent. The cells were pre-treated with PMC (20–100 μM) or an equal volume of solvent control (0.1% DMSO) for 20 min. The cells were treated with a mixture of 50 μg/ml LPS and 100 U/ml IFN-γ (LPS/IFN-γ) or with an equal volume of the solvent control for 24 hrs. The media were collected for nitrite analysis. Nitrite accumulation was determined using the Griess reagent (1% sulphanilamide and 0.1% naphthalenediamine in 2.5% phosphoric acid). The absorbance at 550 nm was measured using an MRX microplate reader (Dynex, Chantilly, VA, USA). Nitrite concentrations were extrapolated from a standard curve constructed using linear regression of the absorbance measurements of sodium nitrite standard solutions prepared using the same culture medium.
6
Quantifying Mouse Alpha-1-Antitrypsin in Lupus
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Mouse AAT levels in lupus mouse serum and pDC culture medium were detected by ELISA. Briefly, pooled B6 (adult male) mouse serum was used as a standard, in which mouse AAT concentration was defined as a one relative unit. Samples (culture medium or sera from lupus mice) and standards were diluted and incubated in a microtiter plate (Immulon 4, Dynex Technologies) in Voller’s buffer overnight at 4°C. Plates were blocked with 3% bovine serum albumin (Sigma) for 1 h at 37°C. Then samples were incubated for 1 h at 37°C. Chicken anti-mouse alpha 1-antitrypsin polyclonal antibody (1:1600 dilution, MyBioscience) and HRP-conjugated goat anti-chicken-IgG antibody (1:5000 dilution, ThermoScientific) were added and incubated for 1 h at 37°C. The plates were washed with PBS-Tween 20 between reactions. After adding substrate (O-Phenyldiamine, Sigma), plates were read at 490 nm on an MRX microplate reader (Dynex Technologies). The OD reading of each sample was used to calculate relative unit based on the standard curve.
7
Quantifying Mouse AAT Levels in Lupus
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Mouse AAT levels in NZM2410 lupus mouse serum were detected by ELISA as previously described.25 (link) Briefly, pooled B6 (adult male) mouse serum was used as a standard, in which mouse AAT concentration was defined as one relative unit. Samples (sera from lupus mice) and standards were diluted and incubated in a microtiter plate (Immulon 4, Dynex Technologies) in Voller’s buffer overnight at 4°C. The plate was blocked with 3% BSA (Sigma) for 1 hr at 37°C. Goat anti-mouse AAT antibody (1:2,000 dilution, MyBiosource, San Diego, CA) was added and incubated for 1 hr at 37°C. Then, horseradish peroxidase (HRP)-conjugated anti-goat-IgG antibody (1:2,500 dilution, R&D Systems, Minneapolis, MN) was added and incubated for 1 hr at 37°C. The plate was washed with PBS-Tween 20 between reactions. After adding substrate (O-Phenyldiamine, Sigma-Aldrich, MO), the plate was read at 490 nm on an MRX microplate reader (Dynex Technologies). The optical density (OD) reading of each sample was used to calculate relative unit based on the standard curve.
8
Cell Viability Assay using MTT
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The viability of cells was assessed by using a colorimetric 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT); (Merck KGaA, Darmstadt, Germany) assay. MTT was diluted with minimum essential medium+Earle's−L-glutamine × 1 (MEM) (GIBCO, Invitrogen) to a concentration of 0.5 mg ml−1. Cells were then incubated with 0.5 mg ml−1 of MTT for 2 h at 37 °C in a humidified atmosphere containing 5% CO2. The solution was then carefully aspirated out and 1 ml of DMSO, (Fisher Scientific, Leicestershire, UK) was added to dissolve the purple-coloured formazan particles and form a homogeneous purple colour. The samples from duplicate wells were transferred to a 96-well plate, and the absorbance of dissolved formazan crystals was spectrophotometrically assessed at a wavelength of 595 nm, using an Mrx microplate reader (Dynex Technologies, Chantilly, VA, USA) and presented in arbitrary units. Viability was defined as the ratio of the optical density of formazan in the treated group against that in the control.
9
Multiplex Cytokine Profiling Assay
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Cytokines IL-1β, IL-4, IL-5, IL-6, IL-10, IL-17A, GM-CSF, IFNγ and TNFα were detected using a custom-made Luminex assay (R&D systems), using the BioPlex 200 system (Bio-Rad, Hercules, CA, USA). Flt3L, IL-23p19 and IL-12p70 were detected by ELISA (R&D systems). Plates were read using an MRX microplate reader (Dyn-ex technologies, Chantilly, VA, USA).
10
Oxaliplatin and Anti-TNF-α Cell Viability
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The CCK-8 kit (Sigma-Aldrich) was used to evaluate cell viability. All procedures were performed according to the manufacturer’s instructions. Briefly, cells (6×103/well) were plated to a 96-well plate with 100 μl culture medium. Gradient concentrations of 10 nM to 104 nM oxaliplatin (Oxa) and anti-TNF-α treatment (infliximab) were added to the medium for culturing for 48 h. After culturing with the CCK-8 solution (10 μl per well) for 30 min at 37°C, the absorbance at 450 nm was read with a MRX microplate reader (Dynex Technologies, USA). The cell viability of each group was determined by their absorbance percentage of the control group.
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