For the determination of intracellular cAMP concentrations, cells were incubated in serum free DMEM (PAA Laboratories, Pasching, Austria) supplemented with 1 mM 3-isobutyl-1-methylxanthine (IBMX; Sigma) for one hour and accumulated cAMP concentrations were determined as previously described by Müller et al. [31] (link). For stimulation curves the medium was supplemented with increasing concentrations of bovine (b) TSH (0–30 mU/ml), recombinant human (rh) TSH (0–100 mU/ml), rhFSH (0–1000 ng/ml) or human (h) FSH analog TR4401 (0–1000 ng/ml). Highly purified bTSH (30 U/mg) was purchased from Dr. A. Parlow and the NIDDK National Hormone and Pituitary Program and reconstituted to 30 U/ml. rhTSH (∼8 U/mg; Thyrogen) was purchased from Genzyme (Neu-Isenheim, Germany) and reconstituted to ∼8 U/ml. rhFSH (44 µg/ml; Gonal-F) was purchased from Merck Serono (Darmstadt, Germany). hFSH analog TR4401 was kindly provided from Dr. Mariusz Szkudlinski (Trophogen, Rockville, Maryland, USA) and reconstituted to 1.1 mg/ml.
Rhfsh
Manufactured by Merck Group
Sourced in Germany, United States
RhFSH is a recombinant human follicle-stimulating hormone (rhFSH) product developed by Merck Group. It is a laboratory equipment used for in vitro research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Gonal f, by Merck Group (3 mentions)
α mem, by Thermo Fisher Scientific (3 mentions)
Activin a, by Merck Group (2 mentions)
Rhfsh, by Promega (2 mentions)
Rhegf, by Promega (2 mentions)
3 isobutyl 1 methylxanthine ibmx, by Merck Group (1 mentions)
Rhtsh, by Sanofi (1 mentions)
Ovitrelle, by Merck Group (1 mentions)
Procrin, by Abbott (1 mentions)
Insulin, by Merck Group (1 mentions)
Transferrin, by Merck Group (1 mentions)
Selenium, by Merck Group (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ksom aa, by Merck Group (1 mentions)
Insulin, by Thermo Fisher Scientific (1 mentions)
Selenium, by Thermo Fisher Scientific (1 mentions)
Transferrin, by Thermo Fisher Scientific (1 mentions)
Catalog 7007, by Corning (1 mentions)
6 protocols using rhfsh
1
Intracellular cAMP Measurement Assay
2
GnRH Agonist Protocol for Controlled Ovarian Stimulation
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The patients were stimulated with a gonadotropin-releasing hormone (GnRH) agonist
protocol (leuprorelin acetate 0.2mL). They were desensitized with 0.2mL per day
of leuprorelin acetate (Procrin, Abbott, Spain) subcutaneously. On the first few
days of menstruation, if baseline levels of estradiol (E2) <50pg/mL were
achieved, ovarian stimulation started with 150-225 of recombinant follicle
stimulating hormone (r-hFSH, Gonal F, Merck Serono, Germany) and 75-150 HP-hMG
(Menopur, Ferring, Switzerland) subcutaneously, per day. Finally, recombinant
human Chorionic Gonadotropin (hCG, Ovitrelle, Merck Serono, Germany) was
administered (250 µg subcutaneously) when at least two follicles had
reached a mean diameter of 17mm. Oocyte retrieval was performed 36h later,
followed by ICSI.
protocol (leuprorelin acetate 0.2mL). They were desensitized with 0.2mL per day
of leuprorelin acetate (Procrin, Abbott, Spain) subcutaneously. On the first few
days of menstruation, if baseline levels of estradiol (E2) <50pg/mL were
achieved, ovarian stimulation started with 150-225 of recombinant follicle
stimulating hormone (r-hFSH, Gonal F, Merck Serono, Germany) and 75-150 HP-hMG
(Menopur, Ferring, Switzerland) subcutaneously, per day. Finally, recombinant
human Chorionic Gonadotropin (hCG, Ovitrelle, Merck Serono, Germany) was
administered (250 µg subcutaneously) when at least two follicles had
reached a mean diameter of 17mm. Oocyte retrieval was performed 36h later,
followed by ICSI.
3
Ovarian Tissue Culture and IVF Protocol
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Ovarian tissue culture was performed using in vitro growth (IVG) medium consisting of αMEM (Invitrogen, CA) supplemented with 5% FBS, 100 mIU/ml rhFSH (MERCK, NJ), 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium (Sigma-Aldrich, MO). Activin A (Sigma-Aldrich, MO) was used as an IVG supplement (30 ng/ml). IVM medium consisted of αMEM supplemented with 5% FBS, 100 mIU/ml rhFSH and 5 ng/ml rhEGF (Promega, WI). TYH medium was used for IVF. After IVF, fertilized oocytes and embryos were cultured in mCZB medium.
4
Cynomolgus Monkey Oocyte Collection and ICSI
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Oocyte collection and fertilization were performed as described previously (Niu et al., 2014 (link)). In brief, 10 healthy female cynomolgus monkeys aged 5–8 years with regular menstrual cycles were selected as oocyte donors for super ovulation, which was performed by intramuscular injection with rhFSH (recombinant human follitropin alpha, Merck, USA) for 8 days, then rhCG (recombinant human chorionic gonadotropin alpha, Merck, USA) on day 9. Oocytes were collected by laparoscopic follicular aspiration 32–35 h after rhCG administration. Follicular contents were placed in Hepes-buffered Tyrode’s albumin lactate pyruvate (TALP) medium (Gibco, USA) containing 0.3% bovine serum albumin (BSA, Sigma, USA) at 37 °C. Oocytes were stripped of cumulus cells by pipetting after brief exposure (<1 min) to hyaluronidase (0.5 mg/mL, Sigma, USA) in TALP-Hepes to allow visual selection of nuclear mature metaphase II (MII; first polar body present) oocytes. The mature oocytes were subjected to intracytoplasmic sperm injection (ICSI) immediately and then cultured in CMRL-1066 containing 10% fetal bovine serum (FBS) (Gibco, USA) at 37 °C in 5% CO2. Fertilization was confirmed by the presence of the second polar body and two pronuclei.
5
Ovarian tissue culture for in vitro growth
6
In Vitro Follicle Culture with NT4
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Basic culture media consisted of αMEM (Gibco, Carlsbad, CA, USA) containing 6% SPS, 1 μl/ml insulin–transferrin–selenium (ITS), 1 mg/ml fetuin, and 10 mIU/ml rhFSH (Gonal-f, MERCK, Darmstadt, Germany). Follicles from each patient were randomly assigned to two study groups and transferred to the corresponding culture media: the control group with basic culture media, and the NT4 group supplemented with 100 ng/ml human NT4 (PeproTech, Rocky Hill, NJ, USA) (Application number of national invention patent of China: 202211330660.7). The chosen concentration of 100 ng/ml was based on a previous study in mice, which was shown to be superior in improving follicle diameter and oocyte maturation in secondary follicle IVG (Guo et al., 2021 (link)). Experiments for the two groups were conducted in parallel. Each follicle was individually incubated in the wells of round-bottom ultra-low attachment microplates (Catalog #7007, Corning, New York, NY, USA) in 200 μl of pre-equilibrated and pre-warmed culture medium at 37 °C and 5% CO2 for 4–6 weeks. Medium refreshment was performed every other day by replacing half of the corresponding culture media. The spent medium was collected and stored at −80°C for subsequent assays.
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