OVCAR-5 and OVCAR-8 cells were seeded at 100,000 cells per well into 12 well plates. After 24 h, cells were treated with 0–50 μM cisplatin in the presence or absence of 20 μM pan-caspase inhibitor Z-VAD-FMK. Negative control wells were treated with PBS. After 2 days of treatment, 5 μCi of [18F]-16 or [18F]-17 in 1 mL serum free media was added per well, followed by 1 h incubation at 37 °C. Cells then were washed 3 × 5 min with 1 mL warmed (37 °C) or cold (4 °C) serum free media. Cells then were lysed by addition of 1 mL of 1N NaOH with 0.5% (w/v) SDS and orbital shaking at 220 rpm for 5 min. Then, 800 μL of lysate was transferred to scintillation vials and decay events measured using a Packard Cobra Quantum Gamma Counter. After decay of the radioisotope, lysate A280 protein concentration was measured using a NanoDrop1000. All CPM data was normalized to the sample protein concentration.
Cobra quantum gamma counter
Manufactured by Hewlett-Packard
The Cobra Quantum gamma counter is a laboratory instrument used to measure the radioactivity of samples. It is designed to detect and quantify gamma radiation emitted by radioactive materials. The core function of the Cobra Quantum is to provide precise and reliable measurements of gamma radiation levels.
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Lab products found in correlation
3 protocols using cobra quantum gamma counter
1
Cisplatin Cytotoxicity in Ovarian Cancer
2
Nanobody-mediated Radioligand Binding Assay
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Competition binding assays (250 µL) contained 60 pM [125I]-CYP, a serial dilution of competitor, the indicated concentration of nanobody/Gs, and approximately 0.5 ng of β2AR nanodiscs diluted in assay buffer (50 mM Tris-HCl pH 7.4, 12.5 mM MgCl2, 2 mM EDTA, 0.05% BSA, 1mM L-ascorbic acid). Total binding was determined in the absence of competitor; nonspecific binding was determined using 10 µM propranolol. Following a 90 min incubation at room temperature, binding assays were terminated by rapid filtration onto GF/B glass-fiber filters treated with 0.3% PEI and washed with 8 mL of cold binding buffer using a harvester (Brandel, Gaitherburg, MD). Bound [125I] was quantified using a Packard Cobra Quantum gamma counter (Packard, San Diego, CA) and expressed as specific binding. For [3H]-ICI 118,551 binding, 10 ng of β2AR nanodiscs were incubated with 0.3 nM radioligand and varying concentrations of nanobody and were harvested as described above. [I125]-CYP affinity was determined using saturation binding (Extended Data Table 1 ). All binding data represent a minimum of three independent experiments with deviation represented as standard error.
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Nanobody-mediated Radioligand Binding Assay
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