Bovine ovaries were collected from a local abattoir and transported to the laboratory (2 h transportation time). Cumulus-oocyte complexes (COCs) were aspirated from all small antral follicles ranging from 2 to 8 mm in diameter. Only COCs covered with at least three layers of cumulus cells were selected and maturated in vitro for 22–24 h. The maturation medium was Tissue Culture Medium 199 (31100-035, Gibco, Grand Island, NY, USA) supplemented with 10% v/v fetal bovine serum (10091148, Thermo Fisher Scientific, Waltham, MA, USA), 100 μM cysteamine (M9768), 0.3 μM sodium pyruvate (P2256), 1% v/v antibiotic-antimycotic (15240-096; Gibco, Grand Island, NY, USA) and 10 μg/mL follicle-stimulating hormone (NIH-FSH-P1; Folltropin ®, Bioniche, Belleville, ON, Canada). COCs were incubated in 100 μL droplets (20–25 COCs/droplet) covered with mineral oil (0121-1; Fisher Chemical, Pittsburgh, PA, USA) at 5% CO2 in humidified air at 38.5 °C. After in vitro maturation, COCs were vortexed for 2 min in 1 mg/mL hyaluronidase (H-4272) in Dulbecco’s phosphate saline (DPBS) for cumulus cell removal. The first polar body extrusion was evaluated by direct observation on a stereoscopic microscope (SMZ 800; Nikon, Tokyo, Japan). Matured oocytes were randomly allocated to activation or IVF groups.
Tissue culture medium 199
Manufactured by Thermo Fisher Scientific
Sourced in United States
Tissue culture medium 199 is a commonly used cell culture medium designed to support the growth and maintenance of a variety of cell types. It provides a balanced combination of amino acids, vitamins, salts, and other essential nutrients required for cell proliferation. This medium is suitable for a wide range of applications in cell biology research and related fields.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Smz800, by Nikon (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Luteinizing hormone, by Merck Group (1 mentions)
Follicle stimulating hormone, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
3 methyladenine 3ma sc 205596, by Santa Cruz Biotechnology (1 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
24 well plate, by Corning (1 mentions)
Stereomicroscope, by Olympus (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
10 protocols using tissue culture medium 199
1
Bovine Oocyte Maturation and Isolation
2
Porcine Oocyte Maturation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All chemicals used in this study were purchased from Sigma-Aldrich, unless otherwise stated. All animal studies were performed after receiving the approval of the Institutional Animal Care and Use Committee (IACUC) of Chungbuk National University, Korea. Porcine ovaries were provided by the local slaughterhouse (Farm story dodarm B&F, Umsung, Chungbuk, Korea) and were transported to our laboratory at 25°C in Dulbecco’s phosphate-buffered saline supplemented with 75 μg/L penicillin G and 50 μg/L streptomycin sulfate. Cumulus–oocyte complexes (COCs) were aspirated from follicles (diameter approximately 2–8 mm) and were washed three times with HEPES-buffered Tyrode’s medium containing 0.1% (w/v) polyvinyl alcohol (PVA)(HEPES is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). The collected COCs were matured in tissue culture medium 199 (Gibco) supplemented with 0.1 g/L sodium pyruvate, 0.6 mM L-cysteine, 10 ng/mL epidermal growth factor, 10% porcine follicular fluid (v/v), 10 IU/mL luteinizing hormone, and 10 IU/mL follicle-stimulating hormone for 44 h at 38.5°C in 5% CO2 and humidified air. After maturation, the cumulus cells were removed by pipetting in the presence of 0.1% hyaluronidase (w/v) for 2–3 min.
3
Porcine Oocyte Maturation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All animal handling and experiments were performed according to a protocol approved by the Animal Research Committee of Chungbuk National University, Korea. All chemicals used in this study were purchased from Sigma-Aldrich, unless otherwise indicated. Porcine ovaries were provided by a local slaughterhouse (Umsung, Cheongju, Korea). Cumulus-oocyte complexes (COCs) were aspirated from the follicles (3–8 mm in diameter) of porcine ovaries and washed three times with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered Tyrode’s medium containing 0.1% (w/v) poly(vinyl alcohol) (HEPES-TL-PVA). The collected COCs were incubated with in vitro maturation (IVM) medium for 44 h at 38.5°C, 5% CO2. The IVM medium comprised tissue culture medium 199 (Gibco) supplemented with 0.1 g/l sodium pyruvate, 0.6 mM L-cysteine, 10 ng/ml epidermal growth factor, 10% porcine follicular fluid (v/v), 10 IU/ml luteinizing hormone (Sigma-Aldrich), and 10 IU/ml follicle-stimulating hormone (Sigma-Aldrich). The COCs were pipetted in TL-HEPES supplemented with 1 mg/ml hyaluronidase (w/v) for 3 min.
4
Porcine Fibroblast Isolation and SCNT
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fibroblast cells were isolated from the ears of four superior purebred Pietrain boars as previously described [24 (link)]. In a humidified atmosphere of 5% CO2 and 95% air, we cultured fibroblasts cells in DMEM (Gibco BRL, Grand Island, NY, USA) supplemented with 10% (v/v) FBS (Gibco BRL, Grand Island, NY, USA) at 39 °C for 8–10 days [25 (link)]. Cultured cells at passages 3–8 were used for SCNT. We collected recipient oocytes from hybrid gilts (Duroc × Yorkshire × Landrace) in a local slaughterhouse. Cumulus oocyte complexes (COCs) were collected from antral follicles and washed with tissue culture medium 199 (Gibco BRL, Grand Island, NY, USA) supplemented with 3.05 mM d-glucose, 0.91 mM sodium pyruvate, 0.1% (w/v) polyvinyl alcohol, 0.57 nM cysteine, 0.5 µg/mL LH, 0.5 µg/mL FSH, 10% (v/v) porcine follicular fluid, 75 µg/mL penicillin G, and 50 µg/mL streptomycin, 10 ng/mL epidermal growth factor [24 (link)]. After incubated for 1.5 days, the oocytes of COCs were released, and matured oocytes with the first polar body (PB) were used for cloning [25 (link)].
5
In Vitro Bovine Oocyte Maturation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ovaries were collected from slaughterhouse and transported to the laboratory. COCs were aspirated from small antral follicles (2-8 mm). COCs covered with at least 3 layers of granulosa cells were selected and maturated in vitro. Maturation medium was Tissue Culture Medium 199 (31100-035, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum, 10 µg/mL follicle stimulating hormone (NIH-FSH-P1; Folltropin ®, Bioniche, Belleville, ON, Canada), 0.3 µM sodium pyruvate (P2256; Sigma-Aldich, St Louis, MO, USA), 100 µM cysteamine (M9768; Sigma-Aldich, St Louis, MO, USA), and 2% antibiotic-antimycotic (15240-096; Gibco, Grand Island, NY, USA). The COCs were incubated in 100 ul droplets (20-25 oocytes/droplet) covered with mineral oil (0121-1; Fisher Chemical, Pittsburgh, PA, USA) for 24 hours at 6.5% CO 2 in humidified air at Revised 38.5°C. Different concentrations of DMSO (D2650; Sigma-Aldich, St Louis, MO, USA) were added to the maturation media, depending on the experiment. After IVM, COCs were vortexed for 2 min in hyaluronidase (H-4272; Sigma-Aldich, St Louis, MO, USA. 1 mg/mL) in Dulbecco´s phosphate saline (to remove cumulus cells). First polar body extrusion was evaluated by direct observation on stereoscopic microscope (SMZ 800; Nikon, Tokio, Japón).
6
Bovine Oocyte Maturation and Autophagy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tissue Culture Medium-199 (TCM-199 containing l -glutamine and phenol red) and fetal bovine serum (FBS) were purchased from GIBCO (Grand Island, NY, USA). Folltropin–V (Follicle-stimulating hormone; FSH) was purchased from Bioniche Animal Health Canada Inc. (Bellevile, Ontario, Canada) and Chorulon (Human chorionic gonadotrophin; hCG) from Intervet Schering Plough (Roseland, NJ, USA). EmbryoMax (KSOM Powdered Media Kit #MR-020P-5F) was purchased from MilliPore (Livingston, Fleming Road, UK). The autophagy inhibitor 3-Methyladenine (3MA; #sc-205596) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The primary antibody [LC3A/B (D3U4C) rabbit monoclonal antibody—#12741] and secondary antibody (anti-rabbit IgG – HRP-linked antibody—#7074) were purchased from Cell Signaling Technology (Danvers, 3 Trash Lane, MA). Frozen semen was purchased from CRV Lagoa (Sertãozinho, São Paulo, Brazil).
7
Porcine Oocyte Maturation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Collecting oocyte complexes (COCs) from porcine ovaries obtained from local
slaughterhouses by aspiration from the follicles (3- to 6-mm diameter) using a
10 mL syringe with 18-gauge needle. After selecting quality COCs, they were
incubated in Tissue Culture Medium 199 (TCM199; Gibco, Grand Island, NY, USA)
supplemented with 0.1% polyvinyl alcohol (PVA), 3.0 mM D-glucose, 0.9 mM
NA-pyruvate, 75 μg/mL penicillin G, 50 μg/mL streptomycin, 0.57 mM
cysteine, 10 μg/mL epidermal growth factor (EGF), 0.01 IU/mL luteinizing
hormone (LH), and 0.01 IU/mL follicle-stimulating hormone (FSH) at 39℃,
5% CO2 in air for 42–44 h.
slaughterhouses by aspiration from the follicles (3- to 6-mm diameter) using a
10 mL syringe with 18-gauge needle. After selecting quality COCs, they were
incubated in Tissue Culture Medium 199 (TCM199; Gibco, Grand Island, NY, USA)
supplemented with 0.1% polyvinyl alcohol (PVA), 3.0 mM D-glucose, 0.9 mM
NA-pyruvate, 75 μg/mL penicillin G, 50 μg/mL streptomycin, 0.57 mM
cysteine, 10 μg/mL epidermal growth factor (EGF), 0.01 IU/mL luteinizing
hormone (LH), and 0.01 IU/mL follicle-stimulating hormone (FSH) at 39℃,
5% CO2 in air for 42–44 h.
8
Porcine Oocyte Maturation with Progesterone and RU486
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Porcine COCs were recovered from follicles 3–6 mm in diameter in porcine ovaries and washed three times with TL-HEPES (with 0.05 g/L gentamycin and 1 g/L polyvinyl alcohol (PVA) added). The collected COCs were matured in IVM medium for 44 h at 38.5 °C in 5% CO2 and humidified air. Normal IVM medium is comprised of tissue culture medium 199 (Gibco) supplemented with 0.1 g/L sodium pyruvate, 0.6 mM l -cysteine, 10 ng/mL epidermal growth factor, 10% porcine follicular fluid (PFF) (v/v), 10 IU/mL LH, and 10 IU/mL FSH. We also tried using normal IVM medium that was not supplemented with PFF, LH, or FSH.
Based on a previous study (Salehnia & Zavareh, 2013 (link); Shimada & Terada, 2002 (link)) different concentrations of progesterone (0 µM, 10 µM, or 100 µM) and RU486 (0 µM, 10 µM, or 25 µM) were added to the culture media. After IVM, the COCs were washed in TL-HEPES (with hyaluronidase (1 mg/mL) and PVA (0.1%, v/v) added) to remove cumulus cells. The oocytes were added to normal TL-HEPES and the oocytes in which the first polar bodies had discharged were selected for further studies.
Based on a previous study (Salehnia & Zavareh, 2013 (link); Shimada & Terada, 2002 (link)) different concentrations of progesterone (0 µM, 10 µM, or 100 µM) and RU486 (0 µM, 10 µM, or 25 µM) were added to the culture media. After IVM, the COCs were washed in TL-HEPES (with hyaluronidase (1 mg/mL) and PVA (0.1%, v/v) added) to remove cumulus cells. The oocytes were added to normal TL-HEPES and the oocytes in which the first polar bodies had discharged were selected for further studies.
9
Ovine Oocyte Maturation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In this study, medium and procedures for oocyte collection and IVM were as described previously (30 (link)). Pre-pubertal ovaries were obtained from a local abattoir and transported to the laboratory in saline at 35–37°C within 2 h. Follicular fluid was aspirated from 3–8 mm antral follicles using a syringe with an 18-gauge needle. Cumulus-oocyte complexes (COCs) in sediments were washed two times in Tyrode's lactate-HEPES-polyvinyl alcohol medium (31 (link)), then selected under a stereomicroscope (Olympus, Tokyo, Japan). After washing three times in IVM medium, 50–70 COCs with uniform oocyte cytoplasm and over 3 layers of compact cumulus cells were cultured in each well of a 24-well plate (Costar, Corning, NY, USA) containing 500 μL IVM medium for 42–44 h at 39°C in an atmosphere of 5% CO2 with saturated humidity. The composition of IVM medium was a tissue culture medium-199 (ThermoFisher Scientific, Grand Island, NY, USA) supplemented with 3.05 mM D-glucose, 0.57 mM cysteine, 0.91 mM sodium pyruvate, 10% (v/v) porcine follicular fluid, 10 ng/mL epidermal growth factor, 0.5 μg/mL each follicle-stimulating hormone, and luteinizing hormone.
10
Porcine Oocyte In Vitro Maturation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
From a local slaughterhouse, porcine ovaries from pre-pubertal gilts were collected and transferred to the laboratory in a saline solution at 32-37°C. The ovarian follicles of 4-8 mm in diameter were aspirated with an 18G needle connected to a 10-ml syringe. The cumulus-oocyte complexes (COCs) were extracted and rinsed three times by using medium comprising 9.5 g/l tissue culture medium-199 (Thermo Fisher Scientific, Waltham, MA, USA), 10 mM N-piperazine-N 0 -[2-ethanesufonic acid] (HEPES), 5 mM sodium hydroxide, 0.3% polyvinyl alcohol (PVA), 2 mM sodium bicarbonate, and 1% penicillin-streptomycin (Invitrogen). After, at least three rinses, the COCs with more than three layers of cumulus cells and homogeneous round cytoplasm were collected and incubated in a 4-well culture dish for 44 h at 39°C under conditions of 5% CO 2 in 95% humidified air. The first IVM medium consisted of TCM-199 liquid form (cat. no. 11150.059), 0.57 mM cysteine, 10 ng/ml epidermal growth factor, human chorionic gonadotropin (hCG), and equine chorionic gonadotropin (eCG) at 10 IU/ml each, 10% porcine follicular fluid, 0.91 mM sodium pyruvate, and 10 μl/ml insulin-transferrin-selenium mixture solution. In addition, after 22 h of maturation, the IVM medium was changed to a hormone-free IVM medium until 44 h.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!