The ovaries were dissociated and placed in precooled PBS solution and as much surrounding tissue was removed as possible. Ovaries were placed in a Transwell chamber (Millicell, Darmstadt, Germany) in Waymouth medium (Sigma, USA) containing 10% (v/v) FBS (Gibco, Staley Rd, Grand Island, NY, USA), 0.23 mM sodium pyruvate, and penicillin and streptomycin (P/S) (Solarbio, Beijing, China) [25 (link)]. For in vitro experiments, ovaries were cultured for 7 days with shRNA or OE-UFL1 lentivirus particle suspension, which was replaced every 24 h.
Waymouth medium
Manufactured by Merck Group
Sourced in Germany
Waymouth medium is a cell culture medium used for the growth and maintenance of various cell types. It provides the necessary nutrients and growth factors required for cell proliferation and survival in vitro. The composition of Waymouth medium is designed to support the specific requirements of the target cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin and streptomycin p s, by Solarbio (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Insulin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Hepes medium, by Thermo Fisher Scientific (1 mentions)
Nu serum 1, by BD (1 mentions)
Dmem ham s f12 medium, by Thermo Fisher Scientific (1 mentions)
Gelatin, by Merck Group (1 mentions)
Nci h295r cells, by American Type Culture Collection (1 mentions)
Pixma mp250, by Canon (1 mentions)
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Collagen 1, by Merck Group (1 mentions)
7 protocols using waymouth medium
1
In vitro Ovarian Culture Protocol
2
Ovarian Tissue Dissociation and Culture
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The ovaries were dissociated and placed in pre-cooling PBS solution and as much surrounding tissue was removed as possible. Ovaries were placed in the Transwell chamber (Millicell, Darmstadt, Germany) in Waymouth medium (Sigma, USA) containing 10% (v/v) FBS (Gibco, Staley Rd Grand Island, NY, USA), 0.23 mM sodium pyruvate, and penicillin and streptomycin (P/S) (Solarbio, Beijing, China).
3
Cell Culture Protocol for Adrenocortical and Leydig Cells
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Human adrenocortical NCI-H295R cells were purchased from American Type Culture Collection (ATCC; CRL-2128). H295R cells were cultured in DMEM/Ham’s F-12 medium containing L-glutamine and 15 mM HEPES medium (GIBCO, Paisley, UK) supplemented with 5% NU-I serum (BD biosciences), 0.1% insulin, transferrin, and selenium (100 U/ml; GIBCO), penicillin (100 U/ml; GIBCO) and streptomycin (100 μg/ml; GIBCO). The serum free starvation medium consisted of DMEM/Ham’s F-12 medium, penicillin and streptomycin (100 μg/ml; GIBCO). Mouse Leydig (MA-10) cells were kindly provided by Prof. Brigitte M. Frey, Bern, Switzerland. MA-10 cells were cultured in Waymouth medium (Sigma–Aldrich) and supplemented with 15% horse serum (GIBCO), penicillin (100 U/ml; GIBCO) and streptomycin (100 μg/ml; GIBCO). For MA-10 cells, culture dishes were pre-coated with 0.1% gelatin (Sigma–Aldrich). The serum free starvation medium of MA-10 consisted of Waymouth medium and antibiotics. Charcoal treatment of NU-I serum was performed by adding charcoal-dextran coated powder (1 g) to NU-I serum (50 mL) while gently mixing on a shaker table overnight. The followingday, charcoal-stripped serum was obtained by centrifugation at 2’000 g for 15 minutes and filtration through a 0.2 μm filter.
4
Collagen Matrix Contraction Assay
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A collagen matrix was set up using 1.8 mg/ml sterile collagen and a neutralization solution (35 (link)). The neutralization solution was made by mixing Waymouth medium (Sigma, W1625) and 2 parts 0.34 M NaOH (Sigma, 221465). One part neutralization mixture was then added to 4 parts of collagen, mixed with 1 × 105 cells to a final volume of 500 μl, and added to each well in a 24-well plate. After a 45-min incubation at 37°C, 1 ml 2% FBS was added to each well, and the plate was incubated for an additional 72 h at 37°C. The medium was then removed, fresh medium and treatment were added, and the collagen matrix was released using a sterile spatula. The plate was scanned using a Canon PIXMA MP250 immediately after release and also at 1, 3, 5, and 24 h. The size of the collagen matrix was measured using ImageJ, and the percentage of contraction was calculated. To decrease any shock to the myofibroblasts, all bacterial strains were grown in DMEM with 2% FBS.
5
Isolation of Primary Murine Hepatocytes
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Primary hepatocytes were isolated from 12-week-old C57BL/6J mice as described in detail previously [24 ]. The cells were plated at 80% confluency on collagen-coated 6-well plates. Collagen-coated plates were prepared at 6.25 μg/cm2 using collagen 1 (Cat#: C3867, Sigma), according to the manufacturer's protocol. Seeded plates were left in the incubator at 37 °C and 5% CO2 for 5 h to allow the cells to attach, then each well was gently washed with 3 mL of Waymouth medium (Cat#: W1625, Sigma) to remove unattached cells and cultured overnight. The medium was then replaced with MEM alpha as indicated above.
6
Ovarian Tissue Culture and Analysis
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Under sterile conditions, mice of different ages were sacrificed and the ovaries were extracted. The ovarian appendage was carefully cleared, and the intact ovaries were rinsed in precooled D-Hanks solution. For ovary culture in vitro, ovaries were placed on a gelatin sponge surface in Waymouth medium (Sigma, USA) containing 10% (v/v) FBS (Gibco, USA), 0.23 mM sodium pyruvate, and 100× penicillin and streptomycin (P/S) (Solarbio, China).
7
In Vitro Ovarian Experiment Protocol
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The ovaries were dissociated and placed in pre-cooling PBS solution and as much surrounding tissue was removed as possible. Ovaries were placed in the Transwell chamber (Millicell, Darmstadt, Germany) in Waymouth medium (Sigma, USA) containing 10% (v/v) FBS (Gibco, Staley Rd Grand Island, NY, USA), 0.23 mM sodium pyruvate, and penicillin and streptomycin (P/S) (Solarbio, Beijing, China) (23) . For in vitro ovarian experiments, ovaries were co-cultured with shRNA or OE-UFL1 lentivirus particles suspension for 7 days which would be replaced every 24h.
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