A 1 mM standard BLM solution was prepared by dissolving an appropriate amount of bleomycin sulfate (Merck, Darmstadt, Germany) in a 0.9% NaCl (saline) solution. This BLM solution was further diluted with saline to obtain BLM solution with a concentration of 10 µM. Sodium acetate and acetic acid used to make an acetate buffer (NaAc—HAc) of pH 4.5, which acted as the supporting electrolyte, were purchased from Merck. There were 1 mM stock solutions of Ni(II), Cd(II), Ca(II), V(V), Fe(III), Mg(II), Cu(II), glucose, ascorbic acid, dopamine, adenine, epinephrine, uric acid and testosterone that were prepared from Merck reagents in deionized water or ethanol (testosterone) and stored at 4 °C in the dark until the influence of interferents was examined. Diethylenetriaminepentaacetic acid (DTPA) was purchased from Merck. Ultra-purified water from a Milli-Q system (Millipore, Livingston, Scotland, UK) was used to prepare the solutions.
Testosterone
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Sao Tome and Principe, Czechia, China, Australia, Italy, Switzerland, Macao, France, Israel, Japan
Testosterone is a laboratory equipment product that measures the concentration of the hormone testosterone in biological samples. It is used in research and clinical settings to assess testosterone levels for various purposes, such as evaluating hormonal imbalances or monitoring treatment effects.
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Lab products found in correlation
Progesterone, by Merck Group (31 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (20 mentions)
Estradiol, by Merck Group (17 mentions)
Diclofenac, by Merck Group (16 mentions)
Nadph, by Merck Group (14 mentions)
Chlorzoxazone, by Merck Group (14 mentions)
17β estradiol, by Merck Group (14 mentions)
Methanol, by Thermo Fisher Scientific (13 mentions)
Cortisone, by Merck Group (13 mentions)
Corticosterone, by Merck Group (13 mentions)
6β hydroxytestosterone, by Merck Group (12 mentions)
Ketoconazole, by Merck Group (12 mentions)
Phenacetin, by Merck Group (12 mentions)
Cortisol, by Merck Group (12 mentions)
Formic acid, by Merck Group (11 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (11 mentions)
Androstenedione, by Merck Group (11 mentions)
Dextromethorphan, by Merck Group (11 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (11 mentions)
Acetonitrile, by Merck Group (10 mentions)
291 protocols using testosterone
1
Preparation and Characterization of Bleomycin Solutions
2
Measurement of Maspin Levels
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Aspirin (acetyl salicylic acid) was purchased from medica zudas health care; I-NAME (NG-nitro-L-arginine methyl ester), Flutamide (2-methyl-N-[4-nitro-3-(trifluoromethyl) phenyl] propanamide), testosterone and estriol were from Sigma Aldrich. Maspin was a gift from Dr. Sally Twining of the Dept. of Biochemistry. Polyclonal maspin antibody was developed in rabbit (13 (link)). testosterone and estriol were purchased from Sigma. Glass microfibre membrane was from Whatman International ltd. Enzyme linked immunosorbent assay (ELISA) maxsorb plate was from Nunc Roskilde. All the chemicals were of analytical grade.
3
Segmental Femur Defect Repair Using AR-Dependent PDCs
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Eight-week-old ARflox/Y or AR-/Y male mice were used to generate a segmental defect of approximately 2.5 mm on the left femur of each mouse using a rotating blade with copious irrigation as we previously described [43 (link)]. Briefly, a 2.5-mm PPF/TCP scaffold loaded with phosphate-buffered saline (PBS; blank) or 100 μg testosterone (Sigma) was inserted to bridge the fracture. After the fracture, 1 × 106 PDCs from ARflox/Y and AR-/Y mice were transplanted into segmental defected mice treated with PBS or testosterone (Sigma). A 25-gauge stainless steel needle was used as an intramedullary pin. The needle was drilled into the trochlear groove between the lateral and medial condyles to reach the femur marrow cavity. It was allowed to pass through the central channel of the scaffold and attached to the proximal end of the femur marrow cavity. Mice were given buprenorphine (0.1 mg/kg) for pain relief for 7 consecutive days, and then were returned to their home cages and allowed to move freely.
4
Granulosa Carcinoma Cell Response
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The experimental set-up consisted of two different granulosa carcinoma cell lines, the KGN cell line and the COV 434 cell line. For each flask, 200,000 cells per flask were cultured in medium DMEM (Gibco, Paisley, UK) supplemented with penicillin/streptomycin (PAA, Cölbe, Germany) and FCS (PAA, Cölbe, Germany). One flask per cell line was treated with one of the following fluids: cell medium only, 50 mM glucose (AppliChem, Darmstadt, Germany), 100 mM glucose (AppliChem, Darmstadt, Germany), DMSO (AppliChem, Darmstadt, Germany), 30 nM testosterone (Sigma Life Science, Steinheim, Germany) or 100 nM testosterone (Sigma, Steinheim, Germany). Cells were harvested after either 24 h or 48 h. All experiments were performed in triplicate.
5
Isolation and Decidualization of Human Endometrial Stromal Cells
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Isolation of human endometrial stromal cells was carried out as described previously.8 Endometrial stromal cells were seeded to 6‐well Costar plates (Sigma‐Aldrich) at a density of 105/well and cultured in DMEM/F12‐Glutamax medium (Thermo Fischer Scientific) supplemented with 10% heat‐inactivated foetal bovine serum (HI‐FBS; Sigma‐Aldrich) and 0.2% penicillin‐streptomycin (Sigma‐Aldrich) until 80%–90% confluency. In vitro decidualization of endometrial stromal cells was induced in phenol red‐free DMEM/F12 (Thermo Fischer Scientific) supplemented with 2% charcoal‐stripped FBS (Sigma‐Aldrich) 0.2% penicillin‐streptomycin (Sigma‐Aldrich) using 1 μM medroxyprogesterone‐17‐acetate (MPA; Sigma‐Aldrich) and 0.5 mM dibutyryl‐cAMP (db‐cAMP; Sigma‐Aldrich) in the presence or absence of 100 nM insulin (Sigma‐Aldrich), 1 μM DHT (Sigma‐Aldrich) or 1 μM testosterone (Sigma‐Aldrich), or the combination of insulin and DHT or insulin and testosterone for six days. The culture media was renewed after 3 days.
In the experiments of flow cytometry, wound‐healing assay and spheroid co‐culture invasion assay, we chose to treat the cells with insulin and/or DHT, but not with testosterone, since DHT has the strongest affinity to the androgen receptor21 and does not convert to other hormones.
In the experiments of flow cytometry, wound‐healing assay and spheroid co‐culture invasion assay, we chose to treat the cells with insulin and/or DHT, but not with testosterone, since DHT has the strongest affinity to the androgen receptor
6
Testosterone-Induced Granulosa Cell Apoptosis
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All animal procedures were approved by the Animal Ethics Committee of Zaozhuang Maternal and Child Health Care Hospital. In total, 84 C57BL/6 mice (weight, 10.3±0.9 g; age, 21 days; total number, 84; immature females) were from Shanghai SLAC Laboratory Animal Co., Ltd. The mice were housed in a temperature (22-24˚C) and humidity (50-60%) controlled environment with a 12-h light/dark cycle and given ad libitum access to food and water. The mice were intraperitoneally injected with 8 IU pregnant mare serum gonadotrophin (Ningbo Sansheng Pharmaceutical Industry Co., Ltd.). At 47 h post-injection, mice were sacrificed by cervical dislocation, the ovaries were collected and the surrounding tissues removed. GCs were isolated from the ovaries by puncturing the follicles with 26-gauge needles, followed by centrifugation at 300 x g for 10 min at 4˚C. GCs were cultured in DMEM/F12 (HyClone; Cytiva) supplemented with 10% FBS (HyClone; Cytiva) and 1% penicillin/streptomycin (HyClone; Cytiva) at 37˚C with 5% CO2. At 1 day post-transduction, cells were incubated with 10 µM testosterone (Sigma-Aldrich; Merck KGaA) or vehicle (0.1% DMSO) for 24 h at 37˚C to induce apoptosis. In addition, cells were treatment with 10 µM testosterone together with 1 µM flutamide (Sigma-Aldrich; Merck KGaA) at 37˚C for 24 h.
7
Steroid Extraction and Quantification
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Pregnenolone (PREG), Pregnenolone-20,21-13C2-16,16 D2 (13C2 D2–PREG), progesterone (PROG), progesterone-2,3,4,20,25-13C5 (13C5–PROG), 17β-Estradiol (17β-E), 17β-Estradiol-2,3,4-13C3 (13C3-17β-E) dihydroprogesterone (DHP), allopregnanolone (ALLO), isoallopregnanolone (ISOALLO), testosterone (T), dihydrotestosterone (DHT), 5α-androstane-3α,17β-diol (3α-diol) and dehydroepiandrosterone (DHEA) were purchased from Merck Life Science, Italy. Acetonitrile, acetic acid, formic acid, methanol, 2-propanol and water were HPLC grade (Merck Life Science, Milano, Italy).
8
In Vitro Transporter Assay Protocol
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Rifampin, diphenhydramide hydrochloride (98%), dimethyl sulfoxide (DMSO), ammonium acetate, acetonitrile Chromasolv (ACN), formic acid, dexamethasone, diclofenac, thiazolyl blue tetrazolium bromide (MTT), and methanol Chromasolv were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bosentan was purchased from Actelion Pharmaceuticals (Allschwil, Switzerland). Cryopreserved human hepatocytes, HEK293 cells overexpressing OATP1B1, OATP1B3, and OATP2B1 transporter, HEK293 mock cells, phosphate-buffered saline (PBS), Gentest High Viability CryoHepatocyte Recovery kit, hepatocyte culture medium, ITS+ culture supplement, Dulbecco’s Modification Eagle’s Medium (DMEM), Minimal Essential Medium (MEM) non-essential amino acid solution, epidermal growth factor (EGF), and Hank’s balanced salt solution (HBSS) were purchased from Corning (Corning, NY, USA). Penicillin/streptomycin, William’s E medium (without phenol red), fetal bovine serum (FBS), l -glutamine, gentamycin sulfate, SYBR Green ER qPCR Supermix, and Superscript First-strand Synthesis kit were purchased from Life Technologies (Burlington, ON, Canada). Sodium butyrate solution and testosterone were purchased from EMD-Millipore (Darmstadt, Germany) and Acros (Geel, Belgium), respectively. Easy Spin Total RNA extraction kit was purchased from iNtRON (Seongnam, Korea).
9
Ellipticine Metabolism Protocol
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Ellipticine, NADH (as disodium salt; purity ~95%), and NADPH (as tetrasodium salt; ~98% purity) were obtained from Sigma Chemical Co (St Louis, MO, USA). Testosterone and 6-β-hydroxyTestosterone were purchased from Merck (Darmstadt, Germany).
10
Enzymatic Inhibitor Binding Kinetics
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All reagents were analytical or HPLC grade. Inhibitors MI-432, MI-463, MI-482 and MI-1900 were synthesized as it has been reported [21] (link). Human serum albumin (HSA), racemic warfarin, CypExpress™ 3A4 Cytochrome P450 human kit, testosterone, 6β-hydroxytestosterone and ketoconazole were purchased from Merck (Darmstadt, Germany). To mimic extracellular physiological conditions, fluorescence spectroscopic measurements were performed in phosphate-buffered saline (PBS; 8.00 g/L NaCl, 0.20 g/L KCl, 1.81 g/L Na2HPO4 x 2 H2O, 0.24 g/L KH2PO4; pH 7.4).
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