72 hours after culture, supernatants were replaced with fresh warm complete media supplemented with 1μCi 3H-thymidine overnight or for a maximum of 18 hours. Incorporated radioactivity was determined by Beta Scintillation Counter (Microbeta Trilux, Perkin Elmer, Wellesley, MA).
Beta scintillation counter
Manufactured by PerkinElmer
Sourced in United States
The beta scintillation counter is a laboratory instrument used to detect and measure the radioactivity of beta particles emitted by a radioactive sample. It functions by converting the kinetic energy of the beta particles into flashes of light, which are then converted into electrical signals and amplified for measurement.
Automatically generated - may contain errors
Lab products found in correlation
3h mpp, by American Radiolabeled Chemicals (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Cd3ε microbeadskit, by Miltenyi Biotec (1 mentions)
3h thymidine, by PerkinElmer (1 mentions)
Ultima gold, by PerkinElmer (1 mentions)
3h cholesterol, by PerkinElmer (1 mentions)
96 well round bottom plate, by Corning (1 mentions)
Phytohemagglutinin (pha), by Roche (1 mentions)
10 protocols using beta scintillation counter
1
Cellular Proliferation Assay Utilizing 3H-Thymidine
2
Evaluating Transporter Inhibition Kinetics
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The medium was removed and immediately replaced with 200 µL/well of Krebs-HEPES buffer (KHB; 10 mM HEPES, 120 mM NaCl, 3 mM KCl, 2 mM CaCl2·2H2O, 2 mM MgCl2·6H2O, 20 mM D-glucose; pH 7.3). The cells were pre-incubated with different concentrations of the drug in KHB at a final volume of 50 µL/well for 5 min (DAT, SERT). Immediately after, the pre-incubation solution was removed and the cells were incubated with the tritiated substrates, 0.02 µM [3H]MPP+ for the DAT (3 min) and 0.1 µM [3H]5-HT for the SERT (1 min), along with different concentrations of the drug in KHB. Finally, the [3H]substrate was removed and the cells were washed with ice-cold KHB followed by the addition of sodium dodecyl sulfate (SDS) 1%. The lysate was then added to the scintillation fluid, and the radioactivity was quantified with a beta-scintillation counter (Perkin Elmer, Waltham, MA, United States).
Non-specific uptake was determined in parallel samples containing cocaine 100 µM for the HEK293-DAT cell line and paroxetine 3 µM for the HEK293-SERT cell line. The non-specific uptake represented <10% of total uptake. The uptake in the absence of the drug compound was normalized to 100%, being the percentage of the uptake in the presence of different concentrations of the drug expressed as a percentage thereof. 3–4 independent experiments were performed in duplicate.
Non-specific uptake was determined in parallel samples containing cocaine 100 µM for the HEK293-DAT cell line and paroxetine 3 µM for the HEK293-SERT cell line. The non-specific uptake represented <10% of total uptake. The uptake in the absence of the drug compound was normalized to 100%, being the percentage of the uptake in the presence of different concentrations of the drug expressed as a percentage thereof. 3–4 independent experiments were performed in duplicate.
3
Cholesterol Synthesis Assay in Cancer Cells
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C4-2 and LNCaP cells were seeded in 12-well plates at a density of 8 × 105 cells per well 72 hours prior to Simvastatin treatment. Following the Simvastatin treatment for 48 hours, the cells were washed twice with HBSS and labeled with 0.5 μCi of 14C-acetic acid prepared in fresh Simvastatin solutions for 3 hours. Cells were washed three times with HBSS following incubation. The lipid content in the samples was extracted using the Bligh Dyer lipid extraction method as previously described, dried under nitrogen, and reconstituted in chloroform [33 (link)]. The chloroform samples were separated using thin layer chromatography on silica gel plate in 9:1 hexane to ethyl acetate solution. A 10 mg/mL cold cholesterol sample and an internal extraction control, 3H [1 (link),2 (link)]-cholesterol loaded at 1 uCi/mL, were included in the thin layer chromatography procedure as controls. After the solution has reached the top, the plate was air-dried and exposed to iodine crystals. The yellow cholesterol bands were excised and reconstituted in scintillation counter fluid to be measured in beta scintillation counter (Perkin Elmer).
4
Radiotracer Uptake Assay for MPP+ in Cells
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On the day of the experiment, cells were incubated with 50 µL Krebs-HEPES-buffer (KHB; 10 mM HEPES, 120 mM NaCl, 3 mM KCl, 2 mM CaCl2, 2 mM MgSO4 and 20 mM D-glucose, pH adjusted to 7.3) containing increasing concentrations of 1-methyl-4-phenylpyridinium (MPP+; Sigma-Aldrich, St. Louis, MO, USA) together with 50 nM [3H]-MPP+ (80–85 μCi mmol−1; American Radiolabeled Chemicals, St. Louis, USA) for 10 min. Time-dependent uptake was determined using 50 nM [3H]-MPP+ for the time indicated. Unspecific uptake was determined in the presence of 100 µM decynium-22. Cells were washed with KHB and then lysed with 100 µL 1% sodium dodecyl sulfate (SDS). Lysate was transferred to a counting vial, containing 2 mL of scintillation cocktail. Uptake of tritiated substrate was assessed with a beta scintillation counter (Perkin Elmer, Waltham, USA).
5
Allogeneic T Cell Proliferation Assay
6
Radioligand Binding Assay of 5-HT Uptake
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The day before the experiment, cells (0.4 × 105/well) were seeded onto PDL (poly-D-lysine)-coated 96 well plates, while excluding the border wells. At the day of experiment the culture medium was aspirated and replaced with Krebs-HEPES buffer (KHB: 120 mM NaCl, 25 mM HEPES, 3 mM KCl, 1.2 mM CaCl2, 1.2 mM MgSO4, 5 mM D-glucose, pH adjusted to 7.3 with NaOH). After 20 min of equilibration the cells were incubated for 10 min with increasing concentration of the compound of interest diluted in 50 µl. Subsequently, tritiated substrate was added (200 nM [3H]5HT). After 1 min, uptake was terminated by aspiration, cell washing with 200 µl cold KHB and lysation with 200 µl of 1 % sodium dodecyl sulphate (SDS). The lysate was mixed with 100 µl scintillation cocktail (Ultima Gold, Perkin Elmer, Waltham, USA) and radioactivity was determined with a beta-scintillation counter (Perkin Elmer, Waltham, USA). Nonspecific uptake was measured in the presence of 10 µM paroxetine and subtracted. Data was plotted by using GraphPad Prism version 9.4.1 (GraphPad Software Inc., San Diego, USA). IC50 values were determined by non-linear regression fits (log(inhibitor) vs. response (Y = Bottom + (Top-Bottom)/(1 + 10^(X-LogIC50)).
7
Uptake Assay for Tritiated Substrate
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Cells were preincubated with vehicle or increasing concentrations of decynium-22 or corticosterone (dissolved in DMSO), diluted in 50 µL of KHB for 10 min. Preincubation solution was aspirated and uptake solution, containing KHB, vehicle or substance of interest at the desired concentration and 50 nM [3H]-MPP+, was added to the wells. Uptake was terminated by aspiration and cell washing with 100 µL ice-cold KHB after 10 min. Cells were subsequently lysed with 100 µL of 1% SDS. Lysate was transferred to a counting vial, containing 2 mL of scintillation cocktail. Uptake of tritiated substrate was assessed with a beta scintillation counter (Perkin Elmer, Waltham, USA).
8
Cholesterol Efflux in M1/M2 Macrophages
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We performed cholesterol efflux assay using [3H]-cholesterol differentiated M1 and M2 macrophages, which were washed with sterile PBS three times and then labeled with 0.5 μCi/mL of [3H]-cholesterol (PerkinElmer, Waltham, MA, USA) in phenol-free RPMI medium and incubated at 37°C for 18 hours. The cells were washed with PBS and equilibrated for 4 hours at 37°C in RPMI medium containing 2% serum from wild type mice as a cholesterol acceptor. The medium was collected and filtered using vacuum filtration system. The cells were lysed in 0.5% SDS for 2 hours at room temperature. Each cell lysate and medium was mixed with scintillation solution and radioactivity of [3H]-cholesterol in each cell lysate and medium was determined by scintillation beta counter (PerkinElmer, Waltham, MA, USA). The cholesterol efflux rate was calculated by disintegration per minute of [3H]-cholesterol in medium/([3H]-cholesterol in medium + [3H]-cholesterol in cell lysate) x 100.
9
Lenalidomide Inhibitory Growth on Myeloma
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The inhibitory growth effect of lenalidomide on myeloma cells was assessed by 3H-thymidine incorporation assay. Briefly, cells were plated in 96-well plates at a density of 5 × 104 cells/well and treated with different concentrations of lenalidomide for 72 hours. 3H-thymidine, 1 μCi, was added to each well, and the cells were incubated for an additional 16 hours. The cells were washed, and the radioactivity was measured using a scintillation beta-counter (PerkinElmer Life and Analytical Sciences, Shelton, CT). The data are expressed as the percentage of the DMSO control value.
10
Amplified T Cell Library Screening
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