MS/MS analysis was performed on an Elute UHPLC interfaced with a Compact Q-TOF (Bruker Daltonics). The nonpolar extract was prepared in MeOH + 0.1% FA (Formic acid) at 10 μg/mL, and a commercial standard of testosterone (T1500, Sigma-Aldrich, St. Louis, MO, USA) was prepared in MeOH + 0.1% FA to 10 mM. Samples (5 µL) were analyzed on a C18 column (C18 Poroshell UPLC 1.9 µm (2.1 × 1.5 mm)) using an isocratic gradient at 60% B (A: H20 + 0.1% FA, B: MeOH + 0.1% FA) over 10 min. The instrument was internally calibrated using ESI Tune Mix (Bruker Daltonics) and set to the following parameters: Mass range: m/z 50–2000, Positive mode, Auto MS/MS with 9 precursor ions per cycle. Testosterone was fragmented at 35 eV.
T1500
Manufactured by Merck Group
Sourced in United States, Germany
The T1500 is a laboratory equipment product manufactured by Merck Group. It is a precision instrument designed for various applications in scientific research and analysis. The core function of the T1500 is to provide accurate and reliable measurements, but a detailed description of its intended use is not available.
Automatically generated - may contain errors
Lab products found in correlation
B6630, by Merck Group (4 mentions)
S3547, by Merck Group (4 mentions)
Elute uhplc, by Bruker (1 mentions)
Esi tune mix, by Bruker (1 mentions)
Compact qtof, by Bruker (1 mentions)
E4250, by Merck Group (1 mentions)
Clozapine n oxide, by Bio-Techne (1 mentions)
E15742 347, by Ssniff (1 mentions)
E2758, by Merck Group (1 mentions)
Gonal f, by Merck Group (1 mentions)
Xbridge c18, by Waters Corporation (1 mentions)
A5040, by Merck Group (1 mentions)
B5002, by Merck Group (1 mentions)
Api 4000, by AB Sciex (1 mentions)
E8875, by Merck Group (1 mentions)
4 androsten 17β ol 3 one, by Merck Group (1 mentions)
Ms 222, by Merck Group (1 mentions)
Hy 101085, by MedChemExpress (1 mentions)
D8418, by Merck Group (1 mentions)
D12451, by Research Diets (1 mentions)
18 protocols using t1500
1
Testosterone Analysis by LC-MS/MS
2
Prenatal Testosterone Exposure in Rats
3
PCOS Model in Pregnant Rats
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Pregnant rats in the PCOS model in the experimental group (n=15) received 0.5 mg/g/day of free testosterone (T1500) (Sigma-Aldrich, Steinheim, Germany) dissolved in a 500 μL cocktail containing sesame oil (S3547) (Sigma-Aldrich, Steinheim, Germany) and benzyl benzoate (B6630) (Sigma-Aldrich, Steinheim, Germany) in a ratio of 4: 1 by subcutaneous injection every day after gestational day 15 until delivery. Pregnant rats in the control group (n=15) received only 500 μL of solvent.
Male and female offspring of the PCOS model (n=12) and the control rats (n=12) were provided ad libitum with food and water. The metabolic phenotype of the offspring and the expression of proteins in white adipose tissue were investigated between 100 and 110 days of age (in adulthood). Histological examination of the ovarian tissue and measurement of serum levels of testosterone in the pregnant rats in the PCOS model were used to confirm the establishment of the PCOS model. Hyperandrogenism was defined as a serum concentration of testosterone <100 ng/ml. Measurement of testosterone was performed according to a standard enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Calbiotech, El Cajon, CA, USA). The pregnant rats that showed no ovarian morphological change or hyperandrogenism were excluded from the study.
Male and female offspring of the PCOS model (n=12) and the control rats (n=12) were provided ad libitum with food and water. The metabolic phenotype of the offspring and the expression of proteins in white adipose tissue were investigated between 100 and 110 days of age (in adulthood). Histological examination of the ovarian tissue and measurement of serum levels of testosterone in the pregnant rats in the PCOS model were used to confirm the establishment of the PCOS model. Hyperandrogenism was defined as a serum concentration of testosterone <100 ng/ml. Measurement of testosterone was performed according to a standard enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Calbiotech, El Cajon, CA, USA). The pregnant rats that showed no ovarian morphological change or hyperandrogenism were excluded from the study.
4
Frog Skin Color Modulation
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After capture, male frogs were placed into black painted plastic boxes (30 cm×30 cm×20 cm) and photographed. Frogs (n = 5 individuals per treatment group) were, 1) injected with hormone (epinephrine or testosterone), 2) injected with a control solution (saline or oil), 3) exposed topically to epinephrine or testosterone, or 4) exposed topically to a control solution. Frogs were injected in the coelomic cavity, at the junction of the underbelly and thigh, away from the vital internal organs, using a thin 1 mL sterile syringe and a sterile 25-gauge needle. For topical treatments, the hormone or control solution was administered onto the dorsal surface of the frog using an eye dropper (same dose as injected frogs). Epinephrine (Sigma-Aldrich E4250) dosage for each frog was 10 µg/0.1 mL 0.9% NaCl (0.0055 M). The testosterone (Sigma-Aldrich T1500) dosage was 1 µg 0.1 mL pure sesame oil solution (0.30–10 M) [25] (link). The controls were 0.1 mL saline (C1) or 0.1 mL oil per frog (C2). Frogs were then photographed at 5, 10, 20, 30, 60 and 120 minutes. Between photographs, a plastic lid was placed on the box so that torch light could not impact skin colour change of the frog [28] .
5
Sex Hormone Replacement and Ablation/Activation in Mice
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Adult mice (8–12 week-old female mice or 14-16 week-old male mice) were bilaterally ovariectomized or castrated. For sex hormone replacement, a 2 cm length of silastic tubing (inner diameter: 1.58 mm; outer diameter: 3.18 mm) containing 36 μg 17β-estradiol/mL (E2758, Sigma)42 in sesame oil or testosterone powder43 (link) (T1500, Sigma) were inserted on the dorsal aspect of the animal’s neck immediately after gonadectomy. Mice were allowed to recover for 1 week before further treatment.
For ablation experiments, 8- to 10-week-old female mice or 14- to 16-week-old male mice were intraperitoneally injected with 20 ng/g bodyweight diphtheria toxin (DT; 322326, EMD Millipore) twice with a 3-day interval between injections. For activation experiments, 2.5 mg of clozapine-N-oxide (CNO; Tocris Bioscience) in 200 ml of drinking water was administered to the mice with a high-fat diet (22% carb, 24% protein, 54% fat, E15742-347, Ssniff). For FSH administration, mice with a high-fat diet received daily 30 IU/kg i.p FSH (GONAL-f, Merck) injections. Mice were weighed and monitored daily before being euthanized as experimental mice. Mice were humanely euthanized if postsurgical complications progressed to a pre-defined humane endpoint at which the mice started to suffer.
For ablation experiments, 8- to 10-week-old female mice or 14- to 16-week-old male mice were intraperitoneally injected with 20 ng/g bodyweight diphtheria toxin (DT; 322326, EMD Millipore) twice with a 3-day interval between injections. For activation experiments, 2.5 mg of clozapine-N-oxide (CNO; Tocris Bioscience) in 200 ml of drinking water was administered to the mice with a high-fat diet (22% carb, 24% protein, 54% fat, E15742-347, Ssniff). For FSH administration, mice with a high-fat diet received daily 30 IU/kg i.p FSH (GONAL-f, Merck) injections. Mice were weighed and monitored daily before being euthanized as experimental mice. Mice were humanely euthanized if postsurgical complications progressed to a pre-defined humane endpoint at which the mice started to suffer.
6
Prenatal Testosterone Exposure and Uterine Contractility
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Seven pregnant rats in the experimental group received 5 mg of free testosterone (T1500; Sigma, Steinheim, Germany) dissolved in a 500μl of sesame oil (S3547, Sigma, Steinheim, Germany) and benzyl benzoate (B6630, Sigma, Steinheim, Germany) with a ratio of 4:1 by subcutaneous injection on the 20th day of pregnancy (16 (link)). A total of 7 pregnant rats in the control group were simultaneously injected with only 500 µL of solvent (sesame oil and benzyl benzoate). After a weaning period, female offspring of experimental and control groups were kept in groups of 4 per cage with ad libitum food and water. Prenatally androgenized (PNA) rats (rat model of PCOS) (n = 7) and controls (n = 7) between 100 - 110 days of age (in adulthood) were assessed for uterine contractility.
7
Isolation of Primary Murine Granulosa Cells
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The isolation of primary granulosa cells was performed as previously reported [55 (link), 56 (link)] with slight modifications. 3-week-old female C57BL/6 mice were euthanized and sterilely dissected to remove the ovaries. The excised ovaries were placed in DMEM/F12 medium containing 1 mg/ml bovine serum albumin and 1% P/S. The tissues surrounding the ovaries were cleaned under a dissecting microscope (Leica, Singapore, M125) and washed twice using the aforementioned medium. GCs were collected by puncturing the excised ovaries with a 25-gauge needle. Cells were resuspended in DMEM/F12 medium (without phenol red) supplemented with 10% charcoal-stripped fetal bovine serum (Vivacell, C3830) and 1% P/S. Cell viability was determined by trypan blue staining, and the purity of granulosa cells was identified using an FSHR antibody (Proteintech, 22665-1-AP). Cells were cultured in basal medium (without phenol red) alone or with different concentrations of testosterone (Sigma, T1500) and FSH for 48 h at 37 °C in a saturated water vapor atmosphere with 95% air and 5% CO2. The culture medium was collected for E2 measurement through an electrochemical method.
8
Testosterone-Filled Silastic Tubing Preparation
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Silastic lab tubing (Catalog number: 508-006, Dow Corning, Midland, MI) with an inner diameter of 1.47 mm and outer diameter of 1.96 mm, was cut into 2-cm pieces and weighed. Silastic tubing was then filled with testosterone (T1500, Sigma) and weighed again to obtain the final dose of 25 mg testosterone within each tube. Tubing was then sealed at both ends with the medical adhesive, acetoxy silicone (A-564, Factor II Inc., Lakeside, AZ), and allowed to dry for at least an hour [17 (link)].
9
Microsomal Stability Assay for CHD1Li
10
Quantifying Testicular Testosterone by LC/MS-MS
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The total testosterone concentration in the testicular culture supernatant of HD and MFD setups was assessed using liquid chromatography/mass spectrometry (LC/MS–MS). The 24-h medium samples were collected into separate tubes on days 7, 28 and 42. The 5-µm-sized LC column (C18, XBridge, WATERS, USA) and testosterone standard (T1500, Sigma-Aldrich, USA) were used for operation. The LC/MS–MS analyses were performed with a Xevo TQ-S spectrometer (WATERS, USA).
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