Striata (inclusive of the dorsal and ventral regions) from adult male mice were dissected and homogenized by tissue tearer and glass homogenizer in homogenization buffer (10 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM EDTA, 1mM DTT), passed through a 26 gauge needle 8 times, centrifuged twice at 20,000 g for 30 minutes at 4°C, and resuspended in assay buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA and 20 μM GDP, 1mM DTT). For each reaction, 2.5 μg of membrane protein were incubated in assay buffer containing ~ 0.1 nM of [35S]GTPγS and increasing concentrations of compounds in a total volume of 200 μL for 2 hours at room temperature. The reactions were terminated by separating membrane bound and free [35S]GTPγS through filtration with GF/B filters using a 96-well plate harvester (Brandel Inc., Gaithersburg, MD). Filters were dried overnight and radioactivity was determined with a TopCount NXT HTS microplate scintillation and luminescence counter (PerkinElmer). Mice were used between the ages of 4–7 months; no apparent changes in receptor coupling were noted in mice from different ages within this range.
Topcount nxt hts
Manufactured by PerkinElmer
The TopCount NXT HTS is a high-throughput scintillation and luminescence detection system designed for microplate-based liquid scintillation counting and luminescence analysis. It features high-speed detection capabilities, automated plate handling, and flexible configuration options to meet the needs of various research and screening applications.
Automatically generated - may contain errors
Lab products found in correlation
Flashplate plus, by PerkinElmer (2 mentions)
Microscint 20, by PerkinElmer (2 mentions)
96 well plate harvester, by Brandel Inc (1 mentions)
Lapatinib, by Chemietek (1 mentions)
Pd173074, by Merck Group (1 mentions)
Vacuum manifold, by Merck Group (1 mentions)
96 well filter plate, by Merck Group (1 mentions)
Pipeline pilot platform, by Dassault Systèmes (1 mentions)
Superdex 200 5 150 gl column, by GE Healthcare (1 mentions)
Taurocholate, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Blasticidin, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
1 2 3h n cholesterol, by PerkinElmer (1 mentions)
7 protocols using topcount nxt hts
1
GTPγS Binding Assay in Mouse Brain
2
Kinase Inhibitor Dose-Response Analysis
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PD173074 (Sigma, St Louis MO) and MK2461 were diluted in DMSO to create a titration series as previously described [14] (link). Gleevec, Lapatinib, PHS665753, and PD168393 were from ChemieTek (Indianapolis IN) Anti IGF1R antibody AF305 was from R/D systems. Cells were treated in triplicate wells, and after 3 days cell numbers were quantitated using the Vialight reagent (Vialight assay kit, Cambrex, Rockland ME). Luminescence was quantified with a Topcount NXT HTS (Perkin Elmer, Waltham, MA) and IC50 determinations made by using logistic 4 parameter curve fitting. For quantitation of phosphotyrosine in FGFR2 and blots were scanned and quantitated using ImageQuant software. Values corresponding to band intensity were plotted against drug concentration to establish an IC50 of drug inhibition. ScanMAX profiling of 442 kinases was at Ambit Biosciences (San Diego, CA).
3
Scintillation Proximity Assay for Methyltransferase Inhibitor Screening
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The assay conditions and protein constructs used in this study are presented in Supplementary Tables 4 and 5 , respectively. The protein purification procedures have been previously described [17 (link), 23 (link), 29 (link), 30 (link)]. In the scintillation proximity assay (SPA), specific amounts of enzyme and biotinylated peptide substrate were mixed with the compound and the reaction was initiated by adding SAM. IC50 values were measured at the apparent Km concentrations of the substrate and SAM. The reaction was quenched by adding an equal volume of 7.5 M guanidine hydrochloride, and the reaction product was measured by SPA using FlashPlate Plus and TopCount NXT HTS plate reader (both from Perkin Elmer Life Sciences).
4
High-Throughput Glucose Uptake Assay
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To measure uptake of glucose in a medium throughput format, 100 μL/well of PBS was added to 96-well filter plates (Millipore) with the Wellmate dispenser. Control wells included either 0 mM (positive control) or 20 mM (negative control) of the competitive inhibitor fructose in PBS. Compounds were added with the Biomek FX automated system. 90 μl of a cell suspension (1.1 x 108 cells/mL) was added to each well with the Wellmate dispenser and left at room temperature for 5 minutes before adding 10 μL of the substrate (4 mM [3H] D-glucose at 50 μCi/mL) to provide a final glucose concentration of 200 μM. After a 5 min incubation, the uptake reaction was stopped by adding 50 μL/well of 4% formaldehyde, followed by incubation for ~5 min. Cells were filtered and washed with a vacuum manifold (Millipore). Plates were dried overnight, 100 μL of scintillation fluid were added, and plates were then sealed and read on a TopCount NXT HTS from PerkinElmer. Data quality and analysis was performed using the GUItars program [22 (link)] and sigmoidal curve fitting with Pipeline Pilot platform (Accelrys, v. 7.0.1).
5
Radiolabeled Cholesterol Binding Assay
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Radiolabeled [1,2-3H(N)]-cholesterol stock in 100% ethanol (PerkinElmer) for each binding reaction (0.5 μM) was aliquoted to glass test tubes. Unlabeled sterol samples in 100% ethanol, used to test competitive inhibition, were then added (20 μM). The solvent was evaporated under a stream of nitrogen gas. Sterols were resolubilized in 120 μl of 25 mM tris (pH 7.4) with 150 mM NaCl and placed in a sonicating bath for 10 min. Ezetimibe series compounds in DMSO were added (20 μM) followed by NPC1L1 protein (0.62 μM). Samples were incubated at 23°C overnight with gentle agitation to allow binding to occur. NPC1L1 protein was then isolated by fast protein liquid chromatography using 100 μl of binding sample fractionated on a Superdex 200 5/150 GL column (GE Healthcare). The column was pulsed with 50 mM taurocholic acid to remove free tracer and reequilibrated after each run. NPC1L1 peak fractions were determined by ultraviolet monitoring, and bound tracer was quantified by liquid scintillation counting of 10-μl fraction with 120 μl of MicroScint-20 (PerkinElmer) and reading the plates on a TopCount NXT HTS (PerkinElmer).
6
Cholesterol Uptake Assay in hNPC1L1/MDCKII Cells
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Madin-Darby Canine Kidney II (MDCKII) cells stably expressing human NPC1L1 (hNPC1L1/MDCKII) were maintained in Dulbecco’s modified Eagle medium (DMEM) (Gibco) containing 1% penicillin/streptomycin (Gibco), 2 mM l -glutamine (Gibco), and blasticidin (5 μg/ml; Invitrogen) supplemented with 5% fetal bovine serum (Gibco). hNPC1L1/MDCKII cells were seeded in 384-well culture plates at a density of 5 × 103 cells per well for a cholesterol uptake assay. The cells were grown at 37°C overnight in a humidified 5% CO2 incubator. Next day, the cell culture medium was completely removed and replaced with DMEM containing test compounds [ezetimibe (Eze), ezetimibe-glucuronide (Eze-Glu), and ezetimibe-PS (Eze-PS); American Radiolabeled Chemicals]. Cells were incubated with compounds at 37°C for 1 hour in a 5% CO2 incubator, followed by addition of 5 nM [1,2-3H(N)]-cholesterol (PerkinElmer) complexed to 10 mM taurocholate (Sigma-Aldrich) in DMEM. After a 90-min incubation for cholesterol uptake, the cells were washed once with phosphate-buffered saline and lysed with MicroScint-20 (PerkinElmer) at room temperature for 1 hour. Radioactivity in the lysate was determined by liquid scintillation counting (TopCount NXT HTS, PerkinElmer).
7
Substrate Preparation and Demethylase Assay
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The ALKBH5 substrate, m 6 A-ss-RNA (5′-UACACUCGA UCUGG(m 6 A)CUAAAGCUGCUC-3′-biotin) was generated using the METTL3-14 complex to completely monomethylate N 6 A ss-RNA. Of the METTL3-14, 500 nM was incubated with 5 µM of biotin-labeled ss-RNA and 5 µM of 3 H-SAM in 20 mM Tris-HCl pH 7.5, 1 mM DTT, 0.01% Triton X-100, 40 U of RNaseOUT/100 µL buffer for 1 h at 23 °C. Then, the reaction mixture was heat shocked at 85 °C for 15 min to denature the methyltransferase METTL3-14 complex, after which the sample was left on ice to cool down. To determine the kinetic parameters, the demethylase assays were performed in 25 nM ALKBH5, 10 µM 2-OG, 100 µM ascorbate, 20 µM Fe (II), and varying concentrations of monomethylated ss-RNA. An equal volume of 7.5 M guanidine-HCl was used to quench ALKBH5 activity after 1 h reaction at 37 °C. Sixty microliters of 20 mM Tris-HCl, pH 8.0, was added into the quenched wells. The quenched reaction mixtures were transferred to a streptavidin/scintillant-coated microplate (FlashPlate Plus, Perkin Elmer Life Sciences), allowed to bind for at least 1 h, and then the decrease of radioactive signals was detected on a Top-Count NXT HTS (Perkin Elmer).
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