A fixed concentration of [3H]DCVJ (10 nM) a pan-amyloid ligand (Kd for α-syn 4.4 nM, Aβ 8.9 nM, tau 15.6 nM), and α-syn (125 nM) prepared as described previously (Paslawski et al., 2016 (link), 2019 (link)) were used with different concentrations of d 2, d 4, d 6, and d 8 from 0.01 to 100 nM. The competitor reaction was diluted with 20 mM Tris-HCl, pH 7.4 to a final volume of 200 μL per well. After incubation for 2 h at 37°C, the binding mixtures were filtered through a Perkin Elmer GF/B glass filter via a TOMTEC cell harvester and immediately washed three times with 1 mL of deionized water. Filters containing the bound ligands were dried and mixed with 3 mL of Betaplate scint solution (PerkinElmer Life Sciences) and incubated for 2 h before counting in a Wallac 1450 MicroBeta TriLux Liquid Scintillation Counter (PerkinElmer Life Sciences). For the determination of the inhibition constants, data were analyzed using GraphPad Prism software (version 7.0) to obtain IC50 values by fitting the data to the equation Y = bottom + (top - bottom)/(1 + 10(x - log IC50). Ki values were calculated from IC50 values using the equation Ki = IC50/(1 + [radioligand]/Kd).
Wallac 1450 microbeta trilux liquid scintillation counter
Manufactured by PerkinElmer
The Wallac 1450 MicroBeta TriLux Liquid Scintillation Counter is a laboratory instrument designed for the detection and measurement of radioactive samples. It utilizes liquid scintillation counting technology to quantify the radioactive emissions from samples.
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Lab products found in correlation
Prism 5, by GraphPad (2 mentions)
Betaplate scint solution, by PerkinElmer (1 mentions)
Cell harvester, by PerkinElmer (1 mentions)
Harvester 96 mach 3, by Tomtec (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Methyl 3h thymidine, by Cytiva (1 mentions)
Meltilex, by PerkinElmer (1 mentions)
Mach 3 cell harvester, by PerkinElmer (1 mentions)
Aim 5 medium, by Thermo Fisher Scientific (1 mentions)
Filter mats, by PerkinElmer (1 mentions)
Cd8 rosettesep, by STEMCELL (1 mentions)
3h thymidine, by PerkinElmer (1 mentions)
6 protocols using wallac 1450 microbeta trilux liquid scintillation counter
1
Competitive Binding Assay for Amyloid Ligands
2
Dopamine Reuptake Inhibition Assay
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A reuptake assay was conducted using HEK-293 cells stably expressing human (hDAT) cDNA. Cells cultured in a 24-well plate were washed with uptake buffer (5 mM Tris base, 7.5 mM HEPES, 120 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl2, 1.2 mM MgSO4, 1 mM ascorbic acid, and 5 mM glucose, pH7.1). The cells were treated with test compounds at 37°C for 20 min, then [3H]-DA was added (final concentration: 20 nM) for 5 min. The cells were washed thrice with ice-cold uptake buffer, and 1% sodium dodecyl sulfate buffer was added into each well overnight. Samples were mixed into a scintillation cocktail, and radioactivity was measured using a Wallac 1450 MicroBeta® TriLux liquid scintillation counter (PerkinElmer Life Sciences). GBR 12909 was the reference compound for DA reuptake inhibition.
Dose-response assays were conducted for each compound to determine the IC50 values. GraphPad Prism 5 (GraphPad, San Diego, CA, USA) was used to analyze data and calculate IC50 values.
Dose-response assays were conducted for each compound to determine the IC50 values. GraphPad Prism 5 (GraphPad, San Diego, CA, USA) was used to analyze data and calculate IC50 values.
3
Determination of IC50 values for BSO, paraquat, and triclosan
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The incorporation of [3H]-hypoxanthine was used to determine IC50 values [42 ] for BSO (L -buthionine sulfoximine), paraquat and triclosan. The starting concentrations for the agents were 1.25 mM for BSO, 0.5 mM for paraquat and 0.4 mM for tricolsan. Following incubation for 48 h, the medium (without further addition of drugs) was replaced and 5 μCi [3H]-hypoxanthine per ml was added to each well. The plates were incubated for a further 24 h after which they were frozen at −20°C. The plates were defrosted at room temperature for 2–3 h before harvesting with a Harvester 96™ Mach III (TomTec) onto Printed Filter Mat A filter mats (Perkin Elmer). These were dried at 55°C for 90 min and sealed into plastic sample bags after addition of 4 ml of scintillation fluid and determining incorporation of [3H]-hypoxanthine using a Wallac 1450 MicroBeta Trilux liquid scintillation counter (Perkin Elmer) for 1 min per well. IC50 values were calculated using GraphPad Prism 5.0.
4
Endocytosis of Dopamine D2 Receptor
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Endocytosis of D2R was determined based on the hydrophilic properties of [3H]-sulpiride (Kim et al., 2001 (link)). HEK-293 cells expressing D2R were stimulated with 10 µM DA for 60 min and incubated with 250 µL of [3H]-sulpiride (2.2 nM, final concentration) at 4°C for 150 min in the presence or absence of an unlabeled competitive inhibitor (10 µM haloperidol). The cells were washed thrice with the same medium, and 1% SDS was added. Samples were mixed with 2 mL Lefkofluor scintillation fluid and counted on a Wallac 1450 MicroBeta TriLux Liquid Scintillation Counter (PerkinElmer Life Sciences).
5
Splenocyte Proliferation Assay for Autoimmunity
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Cell proliferation assays were performed as published previously84 (link). In short, cell suspensions of splenocytes were prepared and the concentration of cells adjusted to 5 × 106 cells/mL. The cells were grown in RPMI-complete medium containing RPMI-1640 (Gibco BRL Carlsbad, CA), 10% FBS, 1x penicillin:streptomycin, 1 mM glutamine, 1 mM nonessential amino acids, and 500 μM 2-ME (Sigma-Aldrich; St. Louis, MO). Splenocytes were stimulated with IRBP161–180 (20 g/mL), S-antigen (concentration), and supernatants of solubilized RPE/choroid or retina extracts for 72 h. For proliferation at 48 hours, 1 Ci of [methyl-3H] thymidine (Amersham Biosciences Pittsburgh, PA) was added to each well of the plate and the mean incorporation of thymidine into DNA was measured at 72 hours by a 1450 Microbeta Wallac Trilux Liquid Scintillation Counter (Perkin-Elmer Life Sciences, Waltham, MA).
6
CD8+ T cell Proliferation Assay
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PBMCs were isolated from 50 healthy donor buffy coats and CD8+ T cells were depleted using CD8+ RosetteSep (StemCell Technologies). Cells were plated at a density of 4–6 million cells per mL in AIM-V medium (Gibco). On days 5, 6, and 7, cells were gently resuspended in 3 × 100 μL aliquots and transferred to each well of a round bottomed 96 well plate. Cultures were pulsed with 0.75 μCi [3H]-Thymidine (Perkin Elmer) in 100 μL AIM-V culture medium, and incubated for a further 18 h, before harvesting onto filter mats (Perkin Elmer), using a TomTec Mach III cell harvester. Counts per minute (Cpm) for each well were determined by Meltilex (Perkin Elmer) scintillation counting on a 1450 Microbeta Wallac Trilux Liquid Scintillation Counter (Perkin Elmer) in paralux, with low background counting.
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