Levonorgestrel (prescribed from Bundang CHA Medical Center) was diluted to four different concentrations (10, 100, 1000, and 10 000 ng/ml) with EGM-2 and introduced into the media reservoir. The drug was dissolved in DMSO at a concentration of 3 mg/mL as a stock solution. Oestradiol (E2; E8875, Sigma, St. Louis, MO, USA) and progesterone (P4; P8783, Sigma) were diluted into 1 μM and 0.1 μM into EGM-2 media, respectively.
P8783
Manufactured by Merck Group
Sourced in United States, Germany
P8783 is a laboratory equipment product manufactured by Merck Group. It is a general-purpose device designed for use in scientific research and analysis applications. The core function of this product is to perform specific tasks or operations required in a laboratory setting. However, a detailed description of its intended use or capabilities is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
M6250, by Merck Group (4 mentions)
H 2270, by Merck Group (3 mentions)
P 7505, by Merck Group (3 mentions)
Gw9508, by Cayman Chemical (2 mentions)
Mmv00, by R&D Systems (2 mentions)
Cck8 reagent, by Zeta Life (1 mentions)
E8875, by Merck Group (1 mentions)
β estradiol, by Merck Group (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Glutamine, by Merck Group (1 mentions)
Transferrin, by Merck Group (1 mentions)
Mitomycin, by Merck Group (1 mentions)
I1882, by Merck Group (1 mentions)
Putrescine, by Merck Group (1 mentions)
M3148, by Merck Group (1 mentions)
M0503, by Merck Group (1 mentions)
F0291, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
12 protocols using p8783
1
Levonorgestrel Dose-Response Assay
2
Progesterone Supplementation in Ovariectomized Rats
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OU rats receiving progesterone were injected subcutaneously with 10 mg/kg progesterone (P8783, Sigma-Aldrich, Germany) diluted in propylene glycol daily for 2 weeks after OVX. Figure 8 shows the protocol of drug administration and surgery procedures. Animals that did not receive progesterone were injected with propylene glycol only (500 μl/kg). The chosen dose of progesterone was based on various studies37 (link),58 (link),59 (link). Plasma progesterone levels were measured using an ELISA kit (Immunotech, Russia).![]()
Progesterone supplementation, experimental design.
3
Cone Photoreceptor Cell Culture and Hypoxia Signaling
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Cone photoreceptor cells47 (link),48 (link) (661W; from Dr. Al-Ubaidi) were cultured as monolayers at 37 °C, 5% CO2 in a humidified atmosphere in DMEM with FBS 10% supplemented with hydrocortisone (20 µg/500 mL, H-2270, Sigma), Progesterone (20 µg/500 mL, P-8783, Sigma), Putrescine (0.016 g/500 mL, P-7505, Sigma) and β-mercaptoethanol (20 µL/500 mL, M-6250, Sigma). Cells were not contaminated by mycoplasma. Equal number of 661W cells (0.3×106) were plated in 6-well dishes and cultured to 80% confluence. Cells were washed twice with PBS, starved for 4 hours (above medium without FBS) then stimulated with GW9508 (14 µM, Cayman) or vehicle. Photoreceptors were then collected 8 hours post-treatment for Hif1a protein expression (see western blot); while their medium was collected at 12 hours for Vegfa quantification by ELISA (as per manual, MMV00, R&D Systems). Vegfa concentration was normalized for the number of cells per well, by doing a Bradford to measure the total cell protein content of each well.
4
Photoreceptor Cell Culture and Gene Knockdown
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Cone photoreceptor cells (al-Ubaidi et al., 1992 (link); Tan et al., 2004 (link)) were cultured as monolayers at 37°C and 5% CO2 in a humidified atmosphere in DMEM with FBS 10% supplemented with 20 µg/500 ml hydrocortisone (H-2270; Sigma-Aldrich), 20 µg/500 ml progesterone (P-8783; Sigma-Aldrich), 0.016 g/500 ml putrescine (P-7505; Sigma-Aldrich), and 20 µl/500 ml β-mercaptoethanol (M-6250; Sigma-Aldrich). No mycoplasma contamination of the cells was detected. An equal number of 661W cells (3 × 105) was plated in each well of 6-well dishes. Cells were treated with AAV2-hRK-sh_control or AAV2-hRK–sh_Vldlr for overnight. On day 2, the cell medium was changed and treated with AAV2-hRK-sh_control or AAV2-hRK–sh_c-fos for overnight. On day 3, the medium was changed with fresh culture medium. After 48 h, photoreceptors were collected to determine gene expression by real time PCR.
5
Cytotoxicity of Niraparib and P4 in Ovarian Cancer
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Ovarian cancer cells were seeded into 96-well plates at 5 × 103 per well, cultured for 24 h, and then treated with vehicle control, niraparib, P4 (P8783, Sigma-Aldrich) plus niraparib or P4 for 48 h. Then, 10 μl of the CCK8 reagent (KOO9-100, Zeta-Life) was added, and cells were incubated at 37 °C with 5% CO2 in the dark for 2 h. Optical density was measured at 450 nm on a spectrophotometer. On the basis of the manufacturer’s instructions, CIs at indicated fraction affected levels were calculated with the CompuSyn software by the Chou–Talalay method with nonconstant ratio combinations. The IC50 and CI values were calculated.
6
Organoid Cultivation with Hormonal Treatments
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Organoids were passaged as described above and allowed to grow for 4 days in standard culture medium (expansion medium; ExM). Afterwards, three different groups were established. Group 1 (ExM) served as an untreated control sample and received ExM for an additional 6 days, with the medium refreshed every other day. Group 2 (E2) was cultured with ExM containing 10 nM beta-estradiol (E8875, Sigma) for 6 days. Again, the medium was renewed every other day. Group 3 (E2+P4) first received ExM supplemented with 10 nM beta-estradiol for 2 days and afterwards ExM with 1 µM progesterone (P8783, Sigma) and 1 µM cAMP (1140, Tocris) in addition to 10 nM beta-estradiol for additional 4 days. After a total of 10 days, all organoids were harvested. For this, medium was removed from the wells and the BME domes incubated with 1× TrypLE Express at 37°C for 10 min. PBS was added to dilute the TrypLE Express and the suspension centrifuged at 478 g for 10 min. After discarding the supernatant, the pellet was resuspended in PBS and centrifuged again to remove leftover BME. After removing the supernatant, cell pellets were snap frozen in liquid nitrogen and then stored at −80°C.
7
Murine CD1 SSC Cell Culture Protocol
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The CD1 SSC cell line from mice was donated by professor Wu Ji’s laboratory from Shanghai Jiao Tong University. The culture medium was based on Minimum Essential Medium α (MEM-α) (12571-063, Gibco, Grand Island, NY, USA) containing 2 mM glutamine (G7012, Sigma, MO, USA), 10% foetal bovine serum (FBS) (16000-36, Gibco), 0.5× pen/strep (15240-062, Invitrogen, Grand Island, NY, USA), 1× nonessential amino acid (NEAA) (11140-050, Gibco) solution, 1× β-mercaptoethanol (β-ME) (M3148, Sigma), 25 μg/ml insulin (I1882, Sigma), 100 μg/ml transferrin (T1428, Sigma), 60 μM putrescine (P5780, Sigma), 60 ng/ml progesterone (P8783, Sigma), 40 ng/ml glial cell line-derived neurotrophic factor (GDNF) (512-GF-050, R&D Systems, Minneapolis, MN, USA) and 3–5 ng/ml basic fibroblast growth factor (bFGF) (F0291, Sigma). The feeder layer cells were STO cells treated with mitomycin (M0503, Sigma). The SSCs were incubated at 37 °C in the presence of 5% CO2.
8
Immunoblotting Protocol for Autophagy
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Whole-cell lysates were prepared in RIPA-Buffer (25 mM Tris-HCl pH 7.6, 150 mm NaCl, 1 mm EGTA, 1% Igepal, 1% sodium deoxycholate, 0.1 % SDS, 1 mm PMSF, 1 mm Na3VO4, 1 mm NaF, 1 μg/mL aprotinin, 1 μg/mL leupeptin, and 1 μg/mL pepstatin). Whole-cell lysates containing 25 μg of whole proteins from lymphoblast cells were loaded for SDS-PAGE electrophoresis. Immunoblots were performed as described in [60 (link)]. The primary antibodies used are described in Supplementary Table S2 . To study autophagy flux, cells were treated with 30 µm hydroxychloroquine (HCQ) for the last 3 h of incubation before lysing. Proteins were extracted with 200 μL lysis Buffer (50 mM Tris-HCl pH 6.8, 10% glycerol (v/v) and 2% sodium dodecyl sulfate (SDS) (w/v) with protease inhibitors 1x (Sigma, P8783), phosphatase inhibitors (1 mm sodium orthovanadate (Sigma, S6508), 1 mm sodium fluoride (Sigma, 201154), and 5 mm sodium pyrophosphate decahydrate (Sigma, 221368)). 20 μg of protein were loaded in CriterionTM TGX Precast Midi Protein gels (BioRad, 5671124) and transferred to PVDF membranes (BioRad, 170–4157). The primary antibodies used are described in Supplementary Table S2 .
9
Assessing Sperm Function with Inhibitors
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Total semen volume, viscosity, pH, appearance, sperm concentration, and motility were assayed after liquefactions with a SQA-VTM sperm quality analyzer (Austria, H18-990). Normal liquefied semen samples were loaded with a sperm medium (Ham’s F-10) and centrifuged at room temperature. The pellets were re-suspended in 0.5 mL of the sperm medium and then incubated for 60 minutes at 37°C under 5% CO2. Motile spermatozoa that progressed from the pellet into the supernatant were collected by aspiration. The number of sperm in suspension was counted using a Neubauer hemocytometer under a light microscope (CX41, Japan). Then according to the number of sperm, Ham’s F-10 was added to adjust sperm concentration to 20×106 sperm/mL. Subsequently, the adjusted sample was divided into 8 experimental groups, containing Ham’s F-10 (control group),1 µM of progesterone (P8783, Sigma Aldrich, Germany), 2 µM of NNC (N0287, Sigma Aldrich, Germany) as CatSper inhibitor, 1 µM of DPI (D2629, Sigma Aldrich, Germany) as NOX5 inhibitor, NNC+DPI, NNC+progesterone, DPI+progesterone, and NNC+DPI+progesterone groups. Thereafter, the samples were incubated at 37 °C and 5% CO2 for 30 minutes. Finally, sperm motility, AR, and viability were evaluated.
10
Tamoxifen Dosing for Pregnant Mice
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We dissolved tamoxifen (T5648; Sigma-Aldrich, St. Louis, MO, USA) in ethanol and then diluted it in corn oil (C8267; Sigma-Aldrich) at a concentration of 10 mg/mL, as described previously32 (link). Then, we injected 1.5 mg or 3 mg of tamoxifen into the peritoneal cavity of pregnant mice at E9 or E15, respectively, and coinjected 1 mg/40 g of progesterone (P8783; Sigma-Aldrich).
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