Both male and female mice were anesthetized with an intraperitoneal (ip) injection of Avertin (0.4 mg 2,2,2-tribronoethanol in 0.02 ml of 2% 2-methyl-2-butanolin saline per gram body weight). For OVX, two 0.5 cm incisions were made bilaterally on the back. A sterile silk ligature was placed around the oviduct, and the ovary and the oviduct were removed through the incisions. For castration, an approximately 0.5–1 cm midline incision was made in the scrotum. A ligation was performed around the vas deferens and spermatic blood vessels and testes were removed. The skin and muscle were sutured. All experiments started 14 days after surgery. The sham mice received the same operation without ligation and removal of ovary and testis. Daily weight measurement over the first week was performed to monitor the health condition. Two weeks after OVX, both CNTF+/+ and CNTF−/− mice were injected subcutaneously with progesterone (10 mg/kg, Cat# 57-83-0, Tocris) for 4 days and the forced swim tests or open field tests were performed 2 h following last progesterone injection.
Progesterone
Manufactured by Bio-Techne
Progesterone is a steroid hormone that plays a crucial role in the female reproductive system. It is primarily produced by the corpus luteum during the luteal phase of the menstrual cycle and by the placenta during pregnancy. Progesterone is essential for the regulation of the menstrual cycle and the maintenance of pregnancy.
Automatically generated - may contain errors
Lab products found in correlation
17β estradiol, by Bio-Techne (4 mentions)
Absolute ethanolvehicle, by Merck Group (2 mentions)
Progesterone, by Agilent Technologies (2 mentions)
17β estradiol, by Agilent Technologies (2 mentions)
Cytation3 spectrophometer, by Agilent Technologies (2 mentions)
Dimethyl sulfoxide (dmso), by Bio-Techne (1 mentions)
Haloperidol, by Merck Group (1 mentions)
Clonazepam, by Merck Group (1 mentions)
Ganaxolone, by Bio-Techne (1 mentions)
Finasteride, by Bio-Techne (1 mentions)
Alphaxalone, by Bio-Techne (1 mentions)
Triazolam, by Merck Group (1 mentions)
Midazolam, by Henry Schein (1 mentions)
8 br camp, by Merck Group (1 mentions)
F9397, by Merck Group (1 mentions)
8 protocols using progesterone
1
Ovariectomy and Castration in Mice
2
Astroglioma Cell-Based CNTF, IL-6, and LIF Expression
3
Pharmacological Manipulation of Hormones
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The following drugs were used: progesterone, DHP, AP (Tocris Bioscience, Bristol, UK), finasteride (Astatech, Bristol, PA) and haloperidol (Sigma-Aldrich, Saint Louis, MO). Finasteride, progesterone, DHP and AP were suspended in 5% Tween 80, diluted with 0.9% saline, and administered by IP injection in a 10 ml/kg volume. haloperidol was dissolved in 10% acetic acid buffered with NaOH and diluted with saline.
4
Steroid and Benzodiazepine Preparation Protocol
5
Bacterial Isolation from Fetal Meconium
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Punch biopsies were taken from three samples of cryopreserved human fetal meconium using a sterile surgical punch biopsy tool (Integra Miltex) in a biosafety class II cabinet. Two independent fetal meconium samples were used for bacterial isolation. Punch biopsies were incubated in pre-reduced liquid brain heart infusion (BHI, TekNova) with 5% defibrinated sheep blood supplemented with 1 × 10−5 M progesterone and 1 × 10−6 M 17β-estradiol (Tocris Bioscience; reconstituted in ethanol) for 48 h at 37°C in an anaerobic chamber under stationary culture conditions. Liquid cultures were streaked onto BHI agar plates with 5% defibrinated sheep blood agar plates to permit individual colonies to be picked and sequenced to assign identity. Colony sequencing (Quintara Biosciences) was performed using the full-length 16S rRNA gene using primer pairs 27 F and 1492 R.54 (link) The full-length 16S rRNA gene was assembled using Clustal Omega, and taxonomy was determined by SINA55 (link) against the curated SILVA database. Additional control strains utilized in our studies were obtained from ATCC (vaginally isolated Lactobacillus iners ATCC# 55195 and Micrococcus luteus ATCC# 4698) or isolated from our previous studies of fetal meconium (M. luteus Micro36 11).
6
Micrococcus growth with sex hormones
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Liquid cultures of Micrococcus strains were grown for
24–48 hours at 37°C in BHI. Cultures were normalized to 0.05
optical density at A600nm (OD600) and incubated with
indicated molar concentrations of progesterone (Tocris Bioscience) and
17β-estradiol (Tocris Bioscience) or equal volume of absolute ethanol
vehicle (Sigma Aldrich), in respective culture media (see above). To test
whether bacterial isolates were capable of growth with progesterone and
17β-estradiol as the sole carbon source, bacterial growth curves were
performed in freshly prepared mineral salt media63 supplemented with
1×10−5M progesterone and
1×10−6M 17β-estradiol or equal volume of
absolute ethanol vehicle at a normalized starting OD600 of 0.1.
Bacterial cultures were then incubated in a Cytation3 spectrophometer (BioTek)
at 37°C for 35 hours, and OD600 was recorded every 15
minutes.
24–48 hours at 37°C in BHI. Cultures were normalized to 0.05
optical density at A600nm (OD600) and incubated with
indicated molar concentrations of progesterone (Tocris Bioscience) and
17β-estradiol (Tocris Bioscience) or equal volume of absolute ethanol
vehicle (Sigma Aldrich), in respective culture media (see above). To test
whether bacterial isolates were capable of growth with progesterone and
17β-estradiol as the sole carbon source, bacterial growth curves were
performed in freshly prepared mineral salt media63 supplemented with
1×10−5M progesterone and
1×10−6M 17β-estradiol or equal volume of
absolute ethanol vehicle at a normalized starting OD600 of 0.1.
Bacterial cultures were then incubated in a Cytation3 spectrophometer (BioTek)
at 37°C for 35 hours, and OD600 was recorded every 15
minutes.
7
Endometrial Stromal Cell Decidualization
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Human endometrial tissue (proliferative phase, n = 20) was obtained from women undergoing surgery for non-malignant gynaecological conditions. None of the women were receiving hormonal therapy or suffering from endometriosis. Cycle phase was determined as previously reported37 (link). Written informed consent was obtained from all subjects prior to surgery, and ethical approval was granted by the Lothian Research Ethics Committee (LREC 10/S1402/59). Methods were carried out in accordance with NHS Lothian Tissue Governance guidelines. Primary endometrial stromal cells (ESC) were isolated from proliferative phase endometrium as described previously37 (link). Decidualization was induced by addition of decidualization media (DM: RPMI 1640, 2% charcoal-stripped FCS, 0.1 mg/ml 8-Br-cAMP (Sigma B5386), 1 μM progesterone (Tocris, Cat no. 2835). Some cells were incubated with the antiandrogen flutamide for the duration of the culture period (10 μM; Sigma F9397). Control cultures were incubated with RPMI 1640, 2% charcoal-stripped FCS and equivalent volume of vehicle control (DMSO). To assess the time-dependent accumulation of secreted products treatments were maintained for the duration of each time point. ESC were treated for 1, 2, 4 and 8 days as indicated.
8
Micrococcus growth with sex hormones
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