Experiments were carried out using the mouse 661W cone photoreceptor-derived cell line, generously provided by Dr Muayyad Al-Ubaidi (Department of Cell Biology, University of Oklahoma, Health Sciences Centre, Oklahoma City, OK, USA). This cell line was previously validated by this group [11 (link)] through real-time quantitative polymerase chain reaction (rt-qPCR) analysis for cone specific opsins; blue cone opsin (Opn1sw) and red/green opsin (Opn1mw). Cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (Sigma) supplemented with 10% fetal calf serum (FCS) and 1% penicillin streptomycin (PS) and maintained at 37°C in a humidified 5% CO2 atmosphere. To analyze the effects of Norgestrel (Sigma) on 661W cells, cells were seeded in to the appropriate culture vessel and allowed to attach overnight. Cells were then washed three times with warmed 1xPBS and complete or serum-free medium supplemented with 20 μM Norgestrel or DMSO vehicle (Sigma) added. After incubation for the indicated times, cells were detached using accutase (Sigma) and, together with their supernatant, centrifuged and washed with 1x PBS to leave a whole cell pellet.
Norgestrel
Manufactured by Merck Group
Sourced in United States
Norgestrel is a synthetic progestin used as a component in certain contraceptive medications. It functions as a hormonal agent that inhibits ovulation and alters the uterine lining to prevent pregnancy. The detailed technical specifications and intended medical applications of Norgestrel are outside the scope of this response.
Automatically generated - may contain errors
Lab products found in correlation
Exemestane, by Merck Group (2 mentions)
Dmso vehicle, by Merck Group (1 mentions)
Dulbecco s modified, by Merck Group (1 mentions)
Accutase, by Merck Group (1 mentions)
Gi254023x, by Merck Group (1 mentions)
Phosphate buffer, by Merck Group (1 mentions)
β glucuronidase, by Merck Group (1 mentions)
Cannabidivarin, by Cerilliant (1 mentions)
Cannabinol, by Cerilliant (1 mentions)
Sesame oil, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Propylene glycol, by Avantor (1 mentions)
Cannabigerol, by Cerilliant (1 mentions)
Cannabidiolic acid, by Cerilliant (1 mentions)
Cannabigerolic acid, by Cerilliant (1 mentions)
Cannabichromene, by Cerilliant (1 mentions)
Tetrahydrocannabivarin, by Cerilliant (1 mentions)
Medroxyprogesterone acetate, by Merck Group (1 mentions)
Norethindrone, by Merck Group (1 mentions)
Luteolin, by Bio-Techne (1 mentions)
9 protocols using norgestrel
1
Norgestrel Effects on 661W Cone Cells
2
Retinal Explant Culture for Neuroprotection
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Retinal explants were cultured from P20 C57 and P15 rd10 mice. Eyes were enucleated and transferred to a sterile laminar flow hood. Whole retinas were carefully dissected and placed, photoreceptor side down, on a cell culture insert in R16 medium supplemented with various other compounds13 (link). Each explant was cultured in one chamber of a 6-well multi-dish in 1.2 ml of medium with 20 μM Norgestrel (Sigma), 100 ng/ml recombinant mouse soluble fractalkine (R&D systems) or vehicle (DMSO or 0.1% BSA in 1x PBS respectively). For experiments using the ADAM10 inhibitor GI254023X (100 nM, Sigma), explants were pre-treated with inhibitor or vehicle (DMSO) for 1 h before the addition of Norgestrel or vehicle (DMSO).
3
Exemestane Quantification by HPLC
4
Exemestane Quantification Method
5
Norgestrel Supplementation in rd10 Mice
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Norgestrel-supplemented and control diets were manufactured by Testdiet (Middlesex, UK). Norgestrel was added to the chow at a concentration of 0.05% (500ppm). Dams of rd10 pups were given a Norgestrel-supplemented diet (LabDiet 5053, custom diet containing D (-) Norgestrel (N2260) (Sigma Aldrich, Missouri, US) when the pups were P10 to allow pups to receive Norgestrel in the milk. This equates to a daily intake of approximately 80mg/kg, assuming a 30g mouse consumes around 5g of food/day. Specialized diet soaked in water was also added to the cage in order for pups to receive Norgestrel directly, as soon as they began to eat. These pups were also given Norgestrel-supplemented diet post-weaning. With control litters, diet changes were replicated with a control diet (LabDiet 5053 control diet). Dams were given regular chow once pups were weaned.
6
Norgestrel Effects on Cone Photoreceptor Cells
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Experiments were carried out using the mouse 661 W cone photoreceptor-derived cell line (passage 25–35), generously provided by Dr Muayyad Al-Ubaidi (Department of Cell Biology, University of Oklahoma, Health Sciences Centre, Oklahoma City, OK, USA). This cell line was previously validated by this group through real time quantitative polymerase chain reaction (rt-qPCR) analysis for cone specific opsins; blue cone opsin (Opn1sw) and red/green opsin (Opn1mw)17 (link). Cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (Sigma) supplemented with 10% fetal calf serum (FCS) and 1% penicillin streptomycin (PS) and maintained at 37 °C in a humidified 5% CO2 atmosphere. To analyze the effects of Norgestrel (Sigma) on 661 W cells, cells were seeded in to the appropriate culture vessel and allowed to attach overnight. Cells were washed with warmed 1xPBS and complete medium supplemented with 20 μM Norgestrel or vehicle (DMSO) was added.
7
Quantitative Analysis of Cannabinoids
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Certified cannabinoid reference standards (at 1 mg/mL in methanol) including cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabigerol (CBG), CBD, tetrahydrocannabivarin, cannabinol (CBN), Δ9-THC, cannabichromene (CBC), cannabidivarin (CBDV), and Δ9-tetrahydrocannabinolic acid-A (THCA) were purchased from Cerilliant (Round Rock, TX). Second-source standards of CBN, CBD, and Δ9-THC were from Restek (Bellefonte, PA). CBD hemp oil was purchased from HempMeds (Poway, CA). Norgestrel, 4-pentylphenyl 4-methylbenzoate (PPMB), ammonium formate, formic acid, and sesame oil were from Sigma-Aldrich (St. Louis, MO). Medium-chain triglyceride (MCT) oil was from Warner Graham (Cockeysville, MD). Propylene glycol was from J.T. Baker (Central Valley, PA). Ammonium formate, formic acid, methanol, acetonitrile, and water were HPLC grade. All other reagents used were analytical grade. Bulk cannabis plant material was obtained from the National Institute on Drug Abuse (NIDA), National Institutes of Health, Bethesda, MD.
8
Hormone Receptor Modulation Protocol
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Luteolin (2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-1-benzopyran-4-one) (LU) was purchased from Tocris (Minneapolis, MN, USA) and dissolved in sterile filtered dimethyl sulfoxide (DMSO; Sigma-Aldrich; St. Louis, MO, USA). Medroxyprogesterone acetate (MPA), progesterone, norethindrone, norgestrel, and RU-486 were purchased from Sigma-Aldrich. Pierce bicinchoninic acid protein reagents were obtained from Fisher Scientific (Pittsburgh, PA, USA). 17-β estradiol (E2; 1.7 mg), MPA (10 mg), and placebo 60-day release pellets were obtained from Innovative Research of America (Sarasota, FL, USA).
9
Norgestrel Exposure in Mosquitofish
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Norgestrel (> 99.9% purity) was purchased from Sigma-Aldrich (St.
Louis, MO, USA). Three nominal concentrations (i.e., 5, 50, and 500 ng L -1 , ∼0.0016, 0.016, and 0.16 mM, respectively) were used in this study. A stock solution (1 mg/mL) for each nominal exposure concentration was prepared by dissolving appropriate amount of NGT in 100% ethanol. Each stock solution was further diluted to the final working concentrations with the exposure medium. For the exposure, adult female mosquitofish were treated with NGT at nominal concentrations of 0 (with ethanol at 0.001%, v/v), 5, 50, 500 ng L -1 . Each treatment had three replicates with 30 fish per replicate. The exposure medium was renewed each day. The experiment was conducted in a semi-static system at 25 ± 1 °C. The water chemistry of the exposure media was maintained similarly as that during the acclimation. The water samples were taken weekly and the NGT concentration in each aquarium was measured (see Section 2.6 for details). The exposure lasted for 42 d.
Louis, MO, USA). Three nominal concentrations (i.e., 5, 50, and 500 ng L -1 , ∼0.0016, 0.016, and 0.16 mM, respectively) were used in this study. A stock solution (1 mg/mL) for each nominal exposure concentration was prepared by dissolving appropriate amount of NGT in 100% ethanol. Each stock solution was further diluted to the final working concentrations with the exposure medium. For the exposure, adult female mosquitofish were treated with NGT at nominal concentrations of 0 (with ethanol at 0.001%, v/v), 5, 50, 500 ng L -1 . Each treatment had three replicates with 30 fish per replicate. The exposure medium was renewed each day. The experiment was conducted in a semi-static system at 25 ± 1 °C. The water chemistry of the exposure media was maintained similarly as that during the acclimation. The water samples were taken weekly and the NGT concentration in each aquarium was measured (see Section 2.6 for details). The exposure lasted for 42 d.
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