3h testosterone

Manufactured by PerkinElmer

Sourced in Italy

[3H]-testosterone is a radiolabeled form of the steroid hormone testosterone, where the hydrogen atoms are replaced with the radioactive isotope tritium (3H). This compound is commonly used in research applications as a tracer to study the metabolism, distribution, and binding of testosterone in biological systems.

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Testosterone (3 citations) Testosterone [1,2,6,7-3H(N)] (2 citations) (1,2,6,7,16,17-3H [N])-Testosterone (2 citations)

6 protocols using 3h testosterone

Lipoprotein Profiling and Hormone Quantification

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Blood was collected in ethylenediaminetetraacetic acid (EDTA) containing tubes. Plasma TAG and cholesterol were measured using colorimetric kits (Infinity). Pooled plasma (100 μL) were separated into lipoproteins using fast‐performance liquid chromatography (FPLC) on a Superose6 column (GE Healthcare, Chicago, IL). We define VLDL as fractions 10–17, LDL as fractions 18–28, and HDL as fractions 30–40. Liver TAG content, liver cholesterol content, and plasma testosterone levels were determined by the Vanderbilt Hormone Assay Core. For liver TAG and cholesterol content, 50–100 mg liver tissue was Folch extracted and separated by thin layer chromatography, which was then analyzed by gas chromatography with internal standards used to control for efficiency of extraction. Testosterone was measured from 25 to 35 μL plasma by radioimmunoassay using [³H]‐testosterone (Perkin Elmer, Waltham, MA). Plasma β‐hydroxybutyrate was measured following 18 h fasting using a colorimetric kit (Cayman, Ann Arbor, MI).

Palmisano B.T., Anozie U., Yu S., Neuman J.C., Zhu L., Edington E.M., Luu T, & Stafford J.M. (2020). Cholesteryl Ester Transfer Protein Impairs Triglyceride Clearance via Androgen Receptor in Male Mice. Lipids, 56(1), 17-29.

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Lipid and Hormone Profiling in Mice

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Blood was collected in EDTA-containing tubes. Plasma TAG and cholesterol were measured using colorimetric kits (Infinity). Pooled plasma (100 μl) were separated into lipoproteins using fast-performance liquid chromatography (FPLC) on a Superose6 column (GE Healthcare). We define VLDL as fractions 10–17, LDL as fractions 18–28, and HDL as fractions 30–40. Liver TAG content, liver cholesterol content, and plasma testosterone levels were determined by the Vanderbilt Hormone Assay Core. For liver TAG and cholesterol content, 50–100mg liver tissue was Folch extracted and separated by thin layer chromatography, which was then analyzed by gas chromatography with internal standards used to control for efficiency of extraction. Testosterone was measured from 25–35 μl plasma by radioimmunoassay using [³H]-testosterone (Perkin Elmer). Plasma β-hydroxybutyrate was measured following 18hr fasting using a colorimetric kit (Cayman).

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Radiolabeled Compound Uptake Assay

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Fetal Bovine Serum (FBS), Penicillin/streptomycin, glutamine, Dextran, Texas Red™ (3000, 10,000, 40,000 and 70,000 MW), B-27™ Supplement (50X), HEPES and Hoechst 33342 were from Invitrogen (Thermo Fisher, Monza, Italy). [¹⁴C]sucrose, [¹⁴C]mannitol, [³H]methyl-glucose, [³H]citalopram, [¹⁴C]leucine, [¹⁴C]phenytoin, [¹⁴C]phenylalanine, [¹⁴C]caffeine, [³H]daunomycin, [³H]testosterone, [³H] [D-Ala2]-Deltorphin II, [³H]propranolol, [³H]verapamil, [³H]prazosin and Microscint 20 Scintillation liquid were purchased from Perkin Elmer, Milan, Italy. [³H]vinblastine, [³H]L-DOPA, [³H]bupropion, [¹⁴C]taxol, [³H]gabapentin, [³H]indomethacin, [³H]raclopride, and [³H]flumazenil are from BIOTREND Chemikalien GmbH (Köln, Germany). Dimethyl sulfoxide (DMSO), lucifer yellow (LY), kynurenic acid, (GF120918), KO143, JPH203, phloretin, and triton X100 were purchased from Sigma-Aldrich, Milan, Italy. Aqueous solutions of 8% paraformaldehyde were obtained from Electron Microscopy Sciences (Hatfield, PA, USA).
Tissue-culture-treated multi-well plates and transwell filter inserts (1.12 cm² growth area, 0.4 µm pore size; transparent polyester) and Corning 96-well plates polystyrene were obtained from Corning (Sigma-Aldrich, Milan, Italy). Optiplate-96 and white Opaque 96-well microplate were from Perkin Elmer, Milan, Italy.

Vignone D., Gonzalez Paz O., Fini I., Cellucci A., Auciello G., Battista M.R., Gloaguen I., Fortuni S., Cariulo C., Khetarpal V., Dominguez C., Muñoz-Sanjuán I, & Di Marco A. (2022). Modelling the Human Blood–Brain Barrier in Huntington Disease. International Journal of Molecular Sciences, 23(14), 7813.

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Detailed Research Reagent Procurement

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Fetal bovine serum, penicillin/streptomycin, glutamine, HEPES, fungizone, deoxyribonuclease I, and Hoechst 33342 were from Invitrogen (Thermo Fisher, Monza Italy). [14C]sucrose, [14C]mannitol, [3H]methyl-glucose, [14C]leucine, [14C]phenytoin, [14C]caffeine, [3H]digoxin, [3H]daunomycin, [3H]testosterone, [3H] [D-Ala2]-deltorphin II, [3H]propranolol, [3H]prazosin and Microscint 20 scintillation liquid were purchased from Perkin Elmer, Milan Italy and [3H]vinblastine, [14C]Taxol, [3H]methotrexate, [3H]gabapentin and [3H]flumazenil were from BIOTREND Chemikalien GmbH (Köln, Germany).
Fluorescein isothiocyanate-labelled FITC-dextran 40, dimethyl sulfoxide (DMSO), lucifer yellow, rhodamine-123, kynurenic acid, elacridar (GF120918), Y-27632 (ROCK inhibitor), and triton X100 were purchased from Sigma–Aldrich, Milan, Italy, and 8% paraformaldehyde aqueous solution was from Electron Microscopy Sciences (Hatfield, PA, USA).
CHDI compounds, denoted cp (A to J) and histone deacetylase (HDAC) inhibitor (cp K) were synthesized as previously described [10 (link),11 (link)].

Di Marco A., Vignone D., Gonzalez Paz O., Fini I., Battista M.R., Cellucci A., Bracacel E., Auciello G., Veneziano M., Khetarpal V., Rose M., Rosa A., Gloaguen I., Monteagudo E., Herbst T., Dominguez C, & Muñoz-Sanjuán I. (2020). Establishment of an in Vitro Human Blood-Brain Barrier Model Derived from Induced Pluripotent Stem Cells and Comparison to a Porcine Cell-Based System. Cells, 9(4), 994.

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Estrogen Receptor Signaling Protocol

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17β-estradiol (E2), ICI 182,780 and DMSO were purchased from Sigma (St. Louis, MO). Triton X, protease inhibitors and LightCycler® 480 SYBR Green I Master were purchased from Roche Molecular Biochemicals (Indianapolis, IN). GPER-1 C-terminal antibody was kindly provided by Dr. Eric Prossnitz, GPER-1 N-terminal antibody was purchased from Abcam, (Cambridge, MA), and Zenon Alexa Fluor Labeling Kit from Molecular Probes, (Eugene, OR). G-1 was purchased from Calbiochem (San Diego, CA) and ³H-testosterone from PerkinElmer Life Science (Boston, MA). MTS Titer Cell Proliferation assay was purchased from Promega Corporation (Madison, WI). Ovine Luteinizing Hormone was provided by the National Hormone and Pituitary Program (NIDDK, Bethesda, MD). Except for estradiol, which was diluted in 30% ethanol:70% Dimethylsulfoxide (DMSO), all ligands were solubilized in DMSO and kept as 1 mM stock solutions at −20°C.

Vaucher L., Funaro M.G., Mehta A., Mielnik A., Bolyakov A., Prossnitz E.R., Schlegel P.N, & Paduch D.A. (2014). Activation of GPER-1 Estradiol Receptor Downregulates Production of Testosterone in Isolated Rat Leydig Cells and Adult Human Testis. PLoS ONE, 9(4), e92425.

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Radioimmunoassay for Steroid Hormones

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Hormone assays were conducted as described by Soma and Wingfield 2001 3 and Wacker et al. 5. Briefly, steroids were extracted from plasma samples with dichloromethane, and DHEA and testosterone were separated under nitrogen pressure on diatomaceous earth/glycol columns and measured via radioimmunoassay, using [³H]‐DHEA (catalogue number NET814250UC; Perkin Elmer, Waltham, MA, USA) or [³H]‐testosterone (catalogue number NET553250UC; Perkin Elmer), respectively 3, 5. All samples were run in duplicate, using polyclonal rabbit anti‐DHEA (catalogue number 20‐DR86; Fitzgerald, Acton, MA, USA) or polyclonal rabbit anti‐testosterone (catalogue number 20R‐TR018W; Fitzgerald) antisera, respectively. The lower detection limits were 0.15 ng/ml for DHEA and 0.29 ng/ml for testosterone. One control value for DHEA and one control value for testosterone were lower than their respective detection limits, and so were assigned the detection limit as their value for calculation of the mean ± SE, as well as for subsequent statistical comparisons. Intra‐assay variations were 16% for DHEA and 9% for testosterone.

Wacker D.W., Khalaj S., Jones L.J., Champion T.L., Davis J.E., Meddle S.L, & Wingfield J.C. (2016). Dehydroepiandrosterone Heightens Aggression and Increases Androgen Receptor and Aromatase mRNA Expression in the Brain of a Male Songbird. Journal of Neuroendocrinology, 28(12), n/a.

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