Radioligand binding was performed as previously described [40 (link),41 (link)]. For the binding assay 50 µL of a dilution series of peptide was added to 50 µL of 3.3 nM [3H]DPDPE (Kd = 3.87 nM) or 2.35 nM of [3H]DAMGO (Kd = 1.07 nM) or 0.8 nM of [3H]U69,593 (Kd = 1.2 nM) in a clear 96 well plate. Next, 100 µL of membrane suspension containing 7 µg protein was added to the agonist wells and incubated for 90 min at room temperature. The reaction mixture was then filtered over a GF-B filter plate (Perkin Elmer) followed by four quick washes with ice-cold 50 mM Tris HCl. The plate was dried overnight, after which 50 µL scintillation fluid (Ultimagold uLLT) was added and radioactivity was counted on a Packard TopCount NXT scintillation counter. All working solutions were prepared in a radioligand assay buffer containing 50 mM Tris HCl, 10 mM MgCl2, and 1 mM ethylenediaminetetraacetic acid at pH 7.4.
Topcount nxt scintillation counter
Manufactured by Hewlett-Packard
Sourced in United States
The TopCount NXT is a scintillation counter designed for the detection and measurement of radioactive samples. It utilizes scintillation technology to convert the energy from radioactive decay into light pulses, which are then detected and quantified. The TopCount NXT provides accurate and reliable measurements of radioactive samples across a wide range of applications.
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12 protocols using topcount nxt scintillation counter
1
Radioligand Binding Assay Protocol
2
Radioligand Binding Assay for Opioid Receptors
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For the binding assay 50 µL of a dilution series of peptide was added to 50 µL of 3.3 nM [3H]DPDPE (Kd = 3.87 nM) or 2.35 nM of [3H]DAMGO (Kd = 1.07 nM) in a clear 96 well plate. Next, 100 µL of membrane suspension containing 7 µg protein was added to the agonist wells and incubated for 90 min at room temperature. The reaction mixture was then filtered over a GF-B filter plate (Perkin Elmer) followed by four quick washes with ice-cold 50 mM Tris HCl. The plate was dried overnight, after which 50 µL scintillation fluid (Ultimagold uLLT) was added and radioactivity was counted on a Packard TopCount NXT scintillation counter. All working solutions were prepared in a radioligand assay buffer containing 50 mM Tris HCl, 10 mM MgCl2, and 1 mM ethylenediaminetetraacetic acid at pH 7.4.
3
Inecalcitol and Palbociclib Proliferation Assay
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BC cells were seeded in 96-well plates and stimulated the next day with a concentration gradient of inecalcitol (10-6 M – 10-13 M) and/or palbociclib (10-5 M – 10-10 M) or a single dose of vehicle (EtOH), inecalcitol (10-8 M) and/or palbociclib (10-7 M). After 72 h of stimulation, 1 µCi [3H]thymidine (specific activity of 2 Ci/mmol, PerkinElmer) was added for an additional 4 h incubation period. Thereafter, cells were harvested on filters and incorporated thymidine was counted using a TopCount NXT Scintillation Counter (Packard).
4
Circadian Rhythm Monitoring in Transgenic Zebrafish Larvae
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Larvae (6 d.p.f.) from a Tg(per3:luc)g1;Tg(elavl3:EGFP)knu3 incross were individually placed into wells of 24-well plates in water containing 0.5 mM beetle luciferin (Promega). From ZT14 (the light to dark transition) the next day, half of the larvae were subjected to a sleep deprivation paradigm (see the ‘Sleep deprivation assay’ section) under dim red light, while the others were left undisturbed in similar lighting conditions. At the end of the 4 h sleep deprivation period, the larvae were individually transferred to the wells of a white-walled 96-round-well plate (Greiner Bio-One) and sealed with an oxygen-permeable plate-seal (Applied Biosystems). Bioluminescence photon counts, reflecting luciferase expression driven by the per3 promoter, were sampled every 10 min for three consecutive days, in constant dark at 28 °C, using the TopCount NXT scintillation counter (Packard).
5
Tissue-Specific Circadian Rhythms in Transgenic Zebrafish
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Adult per3-luciferase transgenic zebrafish (Kaneko and Cahill 2005 (link); Kaneko et al. 2006 (link)) were dissected to investigate direct photoreception in multiple tissues. Specifically, small tissue pieces of brain, fin, gill, gut, liver, muscle, skin, and testis were placed into 96-well dishes, while eye, heart, and pineal and pituitary glands were placed in whole-organ culture and incubated in Leibovitz (L15) media, supplemented with 15% fetal calf serum, penicillin/streptomycin (50 U/mL), gentamicin (50 mg/mL), and 0.5 mM beetle luciferin (Promega). Subsequently, plates were exposed for up to 6 d to two different lighting regimes comprising (1) a forward light cycle consisting of 12 h of light, followed by 12 h of dark (LD), or (2) a reverse light cycle comprising 12 h of dark, followed by 12 h of light (DL), before being transferred into constant darkness (DD) for up to 7 d. A bioluminescence reporter assay measured gene activity during the entire 2 wk of the experiment (LD or DL into DD) on a Packard TopCount NXT scintillation counter at 28°C.
6
Zebrafish Bioluminescence Assay Protocol
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For all experiments, single embryos were aliquoted into individual wells of a 96-multiwell plate (Nunc) in E3 media (without Methylene Blue) supplemented with 0.5 mM beetle luciferin, potassium salt solution (Promega) and the plate was then sealed using an adhesive “Top Seal” sealing sheet (Packard). Plates were then subjected to different temperature or lighting conditions and bioluminescence from whole embryos and larvae was assayed using a Packard Top-count NXT scintillation counter (2-detector model, Packard) or an EnVision multilabel counter (Perkin Elmer). Bioluminescence data was analyzed using the Import and Analysis Macro (I&A, Plautz and Kay, Scripps) for Microsoft Excel or CHRONO software [28 (link)].
7
Chromium-Release Cytotoxicity Assay for Tumor Cell Lines
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Tumor cell lines were labeled with 51Cr (Perkin Elmer) for 2 hours at 37°C; then washed twice with 2% FBS in PBS and once with X-VIVO 20 media containing 5% human AB serum. T cells were plated with the target cells in triplicate at varying effector-to-target (E:T) ratios and incubated for 4 hours at 37°C. Following incubation, 30 uL aliquots of supernatant were collected into 96-well LumaPlates (Perkin Elmer) and read using a TopCount NXT Scintillation Counter (Packard) or Microbeta A2450-0020 Plate Reader (Perkin Elmer). Spontaneous release was measured by incubation of target cells with media alone. Maximum release was achieved by lysis of target cells with a 0.05% Triton-X solution. Specific lysis was calculated using the formula: % specific lysis = [(experimental release - spontaneous release)/(maximum release - spontaneous release)] x 100. Triplicate values were averaged prior to calculation of the specific lysis; data from multiple experiments were averaged.
8
Circadian Rhythms in Transgenic Zebrafish
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Total bioluminescence of brain tissue dissected from per3-luciferase transgenic zebrafish was monitored using a Topcount NXT scintillation counter (Packard) at 28°C. Tissues were dissected and placed into 96-well plates in L15 media described above containing 0.5 mM luciferin. The lighting conditions are indicated on the figures and described in the figure legends. To determine period and phase, these values were standardised with splinefun function (http://bitly.com/Sfse6r ) from the statistics package for R (http://www.r-project.org/ ). Measurements were smoothed according to a kernel regression using the npreg (http://bitly.com/VlENLI ) regression from the np package for R, with a bandwidth chosen by expectation maximization. Finally, the first derivative points of the signals gave the time of the peak and troughs throughout the recording, from which the period length and phase shifts were measured.
9
Quantifying PP2A Methylation in 96-Well Plate
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Example 23
Measuring PP2A Methylation in 96-Well Plate Format
The present example demonstrates that compounds of the present invention modulate the methyltransferase activity [i.e., the carboxyl methylating activity of the protein phosphatase 2A specific methyltransferase (MTase)], wherein such activity results in PP2A methylation. Purified PP2A, MTase and [3H]-SAM, were incubated in reaction buffer at 37° C. At the end of the reaction, mixture was added onto the membranes of 96-well filter plate (Multiscreen™, Millipore). The membranes were then pre-wetted with 70% ethanol (50 μL/well) and then subsequently washed with water (2×200 μL/well). The reaction in each well was terminated and the proteins (5 μL-20 μL) were precipitated by adding 25% ice-cold TCA. The plate was kept on ice for 30 minutes to ensure completion of protein precipitation. Excess of the free SAM was then removed by washing with 5% cold TCA (50 L/well) and 70% cold ethanol (2×100 μL/well). Membranes were air-dried, radioactivity in each well was counted by TopCountNXT scintillation counter (Packard) with 25 μL/well of Microsinct™ 20 (PerkinElmer). Exemplary results are presented in Table 2 below.
10
Kinetics of CGRP Receptor Antagonist Binding
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Association kinetic assays were performed by combining 40 pM [ 3 H]-ubrogepant with 50 mg SK-N-MC membranes per well in binding buffer and incubating at room temperature for various times (1-90 minutes). Dissociation kinetic assays were performed by combining 40 pM [ 3 H]-ubrogepant with 50 mg SK-N-MC membranes per well in binding buffer and incubating at room temperature for 3 hours. At that point, 10 mM of the CGRP receptor antagonist MK-3207 was added and dissociation was monitored for various intervals (1-300 minutes). All assays were terminated by filtration through 0.5% polyethyleneimine-treated GF/B glass fiber plates with ice-cold wash buffer (10 mM HEPES, pH 7.4, and 5 mM MgCl 2 ). Plates were dried under vacuum at 37°C for 1 hour, scintillation fluid was added, and radioactivity quantitated using a Packard TopCount NXT scintillation counter.
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