Oocytes were aspirated from antral follicles (2–4 diameters) of ovaries from prepubertal gilt ovaries collected at a local slaughterhouse (Olson Meat Company, Orland, CA, USA). COCs were washed in TCM-199 (GIBCO) containing 0.1% polyvinyl alcohol (PVA), and incubated at 38°C and 5% CO2 for 48 hr in TCM-199 containing 0.1% PVA, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.5 μg/ml oFSH, 0.5 μg/ml bLH, 10 ng/ml EGF, 10 μg/ml gentamicin (GIBCO) and 10% porcine follicle fluid.
Tcm 199
Manufactured by Thermo Fisher Scientific
Sourced in United States, Spain, Canada, Denmark, Belgium, Japan
TCM-199 is a cell culture medium developed for the in vitro cultivation of various cell types, including mammalian cells. It provides the necessary nutrients and growth factors to support cell growth and maintenance. The formulation is designed to maintain the physiological pH and osmolality required for optimal cell performance.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (15 mentions)
Bovine serum albumin (bsa), by Merck Group (7 mentions)
Gentamicin, by Thermo Fisher Scientific (6 mentions)
Four well plate, by Thermo Fisher Scientific (5 mentions)
Cysteamine, by Merck Group (5 mentions)
Tcm 199, by Merck Group (5 mentions)
4 well dish, by Thermo Fisher Scientific (4 mentions)
Four well dish, by Thermo Fisher Scientific (4 mentions)
Epidermal growth factor (egf), by Merck Group (4 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (4 mentions)
Pen strep, by Thermo Fisher Scientific (4 mentions)
Humid incubator, by Astec (3 mentions)
Sodium pyruvate, by Merck Group (3 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (3 mentions)
Mineral oil, by Nacalai Tesque (3 mentions)
Paraffin liquid, by Nacalai Tesque (3 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (3 mentions)
17β estradiol, by Merck Group (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (3 mentions)
Pregnant mare serum gonadotropin, by Lee Biosolutions (3 mentions)
173 protocols using tcm 199
1
Porcine Oocyte Maturation Protocol
2
Porcine Oocyte Maturation in Vitro
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Oocytes were aspirated from antral follicles (2–4 diameters) of ovaries from prepubertal gilt ovaries collected at a local slaughterhouse (Olson Meat Company, Orland, CA, USA). COCs were washed in TCM-199 (Gibco) containing 0.1% polyvinyl alcohol (PVA), and incubated at 38 °C and 5% CO2 for 48 hours in TCM-199 containing 0.1% PVA, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.5 μg/ml oFSH, 0.5 μg/ml bLH, 10 ng/ml EGF, 10 μg/ml gentamicin (Gibco) and 10% porcine follicle fluid.
3
Goat Embryo Nuclear Transfer Protocol
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The qualified oocytes were transferred into TCM199 (Gibco, Brooklyn, NY, USA) with 2% (v:v) FBS. Mature oocytes were incubated in TCM199 medium (Gibco, Brooklyn, NY, USA) supplemented 5 μg/mL CB (Sigma-Aldrich, St. Louis, MO, USA; C6762) and 5 μg/mL Hochest 33,342 (Beyotime, Tongzhou, Beijing, China, C1022) about 5–10 min. Then, with the help of a micromanipulator, mature oocytes were enucleated and then rhBChE-positive GFFs were injected into the enucleated oocytes. After 90 min, these oocytes were performed electric fusion. Firstly, injected oocytes were transferred into a fusion solution for 3–5 min. Then, oocytes were placed into a fusion tank covered with the fusion solution.
The polar body of the oocyte was oriented parallel to the electrode and a DC pulse was applied 2 direct current pulses of 2.0 kV/cm for 25 μs using an ECM2001 Electrocell Manipulator (BTX Inc., San Diego, CA, USA). The fused embryos were transferred into TCM199 medium including 10 μg/mL of actinomycetes CHX (Sigma-Aldrich, St. Louis, MO, USA, C7698) and 5 μg/mL of CB for 4–5 h. Finally, the embryos were incubated to the developmental solution and transplanted the next day.
Sixty days after the embryo transfer, we would assess the pregnancy rate of the female goats via ultrasonography.
The polar body of the oocyte was oriented parallel to the electrode and a DC pulse was applied 2 direct current pulses of 2.0 kV/cm for 25 μs using an ECM2001 Electrocell Manipulator (BTX Inc., San Diego, CA, USA). The fused embryos were transferred into TCM199 medium including 10 μg/mL of actinomycetes CHX (Sigma-Aldrich, St. Louis, MO, USA, C7698) and 5 μg/mL of CB for 4–5 h. Finally, the embryos were incubated to the developmental solution and transplanted the next day.
Sixty days after the embryo transfer, we would assess the pregnancy rate of the female goats via ultrasonography.
4
Oocyte Maturation and Proteomic Analysis
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Oocyte collection and maturation were done as described [35 (link)]. Briefly, immature oocytes were aspirated with a syringe from follicles 2–6 mm in diameter, from abattoir-derived ovaries. Immature oocytes were washed in TCM199 (Gibco, Grand Island, NY, USA) supplemented with 0.3% bovine serum albumin and once more in maturation medium: TCM199 supplemented with 5% estrous bovine serum and 10 µg/mL FSH. They were cultured in sterile mineral oil at 38.5 °C under 5% CO2 in humidified air for 22–24 h. Following maturation, the oocytes with expanding cumulus cells were selected for proteomic analysis. The cumulus cells were stripped off by repeated pipetting.
5
Porcine Oocyte Maturation with Ramelteon
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Porcine ovaries were placed into a thermos and transported to the laboratory at 30–34 °C from the local slaughterhouse, and the 3–6 mm diameter follicles were aspirated using a syringe with an 18-guage needle. Cumulus-oocyte complexes (COCs) were collected with several cumulus cells layers, and washed 3 times with tissue culture medium-199 (TCM-199; Invitrogen, Carlsbad, CA, USA) containing 10 mM HEPES, 1% penicillin-streptomycin and 0.3% polyvinyl alcohol (PVA); Finally, COCs were placed into IVM medium (TCM-199 containing 0.91 mM sodium pyruvate, 10% porcine follicular fluid, 10 IU/mL follicle stimulating hormone and 10 IU/mL luteinizing hormone). Selected 50 COCs/condition (with 0, 10−11, 10−9, 10−7, and 10−5 M ramelteon) was then cultured in an incubator at 5% CO2, and 38.5 °C for 42 h.
6
Porcine Oocyte In Vitro Maturation
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Porcine ovaries were maintained at 30–37 °C during transportation, and small antral follicles (diameter: 3–6 mm) were aspirated using a syringe. COCs, with numerous layers of cumulus cells, were collected; washed using tissue culture medium-199 (TCM-199; Invitrogen, Carlsbad, CA, USA) containing 0.3% polyvinyl alcohol (PVA), 10 mM HEPES and 1% penicillin–streptomycin; and transferred into culture dishes containing IVM medium (TCM-199 containing 10 IU/mL luteinizing hormone, 10 IU/mL follicle stimulating hormone, 10% porcine follicular fluid and 0.91 mM sodium pyruvate). Pooled 50 COCs/condition was then cultured in an incubator at 5% CO2, 100% RH and 38.5 °C for 42 h (with or without 10−9 M MTn or 10−9 M MTn antagonist).
7
Porcine Oocyte In Vitro Maturation
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Ovaries were obtained from prepubertal gilts at a local slaughterhouse, and were transported to the laboratory within 3 h after collection in physiological saline at 32–35 °C. Cumulus oocyte complexes (COCs) were aspirated from antral follicles (3 to 6 mm in diameter) using an 18-gauge needle fixed to a 10 mL disposable syringe, and allowed to settle in 50 mL conical tubes at 37 °C for 5 min. The supernatant was discarded and the sediment was washed three times in washing medium comprising tissue culture medium (TCM)-199 (Invitrogen, Carlsbad, CA, USA) containing 5 mM sodium hydroxide, 2 mM sodium bicarbonate, 10 mM HEPES, 0.3% polyvinyl alcohol, and 1% Pen-Strep (Invitrogen). In each experimental group, approximately 50 COCs were placed into an IVM medium comprising TCM-199 supplemented with 10 ng/mL epidermal growth factor, 0.57 mM cysteine, 0.91 mM sodium pyruvate, 10 μL/mL insulin transferrin selenium solution (ITS-A) 100× (Invitrogen), 10% porcine follicular fluid, 10 IU/mL eCG, and 10 IU/mL hCG. The COCs were cultured at 39 °C in a humidified atmosphere of 5% CO2. After 21–22 h of maturation culture with hormone, the COCs were washed three times in fresh hormone-free IVM medium, and then cultured in hormone-free IVM medium for another 21–22 h.
8
Isolation and Culture of Bovine Oocytes
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After transport from the slaughterhouse to the laboratory at 30℃ (within 0.5-1 h), ovaries were cleaned by trimming the adnexa with scissors and washing with sterile saline. All visible follicles were opened with a scalpel and the follicular wall was scraped using a bone curette. Scraped contents were washed into petri dishes with D-MEM/F-12 supplemented with 0.05% (w/v) polyvinyl alcohol and examined under a stereomicroscope. Cumulus-oocyte complexes were classified and held in a mixture of 40% (v/v) TCM-199 with Earle's salts (Invitrogen) and 40% (v/v) TCM-199 with Hanks' salts (Invitrogen) with 20% (v/v) FBS [20 (link)] at room temperature for 23 h (holding treatment). After the holding treatment, COCs were washed with fresh IVM medium and cultured at 38.5℃ in a humidified atmosphere of 5% CO2 in air for 19 to 20 h.
9
Porcine Oocyte In Vitro Maturation
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Pig ovaries were obtained from prepubertal gilts at a local slaughterhouse and transported to the laboratory within 3 h in sterile saline at 32–35°C. Cumulus oocyte complexes (COC) were
collected from follicles of 3–6 mm diameter by aspiration with an 18-gauge needle. Oocytes with homogeneous ooplasm surrounded by several compact layers of cumulus cells were selected and
washed three times with tissue culture medium-199 (TCM-199; Invitrogen, Carlsbad, CA, USA) containing 5 mM sodium hydroxide, 2 mM sodium bicarbonate, 10 mM N-[2-Hydroxyethyl]
piperazine-N′-[2-ethanesulfonic acid] (HEPES), 0.3% polyvinyl alcohol (PVA), and 1% Pen-Strep (Invitrogen). The COCs were placed into in vitro maturation medium (IVM)
containing TCM-199 supplemented with 0.57 mM cysteine, 0.91 mM sodium pyruvate, 5 μl/ml insulin-transferrin-selenium solution (ITS-A) 100X (Invitrogen), 10 ng/ml epidermal growth factor
(EGF), 10% porcine follicular fluid (v/v), 10 IU/l equine chorionic gonadotropin (eCG), and 10 IU/ml human chorionic gonadotropin (hCG), and incubated at 38.5°C under 5% CO2 in
95% humidified air. Following 21–22 h of maturation with hormones, the COCs were washed twice in fresh, hormone-free IVM medium and then cultured for an additional 21–22 h.
collected from follicles of 3–6 mm diameter by aspiration with an 18-gauge needle. Oocytes with homogeneous ooplasm surrounded by several compact layers of cumulus cells were selected and
washed three times with tissue culture medium-199 (TCM-199; Invitrogen, Carlsbad, CA, USA) containing 5 mM sodium hydroxide, 2 mM sodium bicarbonate, 10 mM N-[2-Hydroxyethyl]
piperazine-N′-[2-ethanesulfonic acid] (HEPES), 0.3% polyvinyl alcohol (PVA), and 1% Pen-Strep (Invitrogen). The COCs were placed into in vitro maturation medium (IVM)
containing TCM-199 supplemented with 0.57 mM cysteine, 0.91 mM sodium pyruvate, 5 μl/ml insulin-transferrin-selenium solution (ITS-A) 100X (Invitrogen), 10 ng/ml epidermal growth factor
(EGF), 10% porcine follicular fluid (v/v), 10 IU/l equine chorionic gonadotropin (eCG), and 10 IU/ml human chorionic gonadotropin (hCG), and incubated at 38.5°C under 5% CO2 in
95% humidified air. Following 21–22 h of maturation with hormones, the COCs were washed twice in fresh, hormone-free IVM medium and then cultured for an additional 21–22 h.
10
Porcine Oocyte Isolation and In Vitro Maturation
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Porcine ovaries were recovered from a local abattoir, placed in saline solution at 30℃–35℃, and transported to the laboratory. Follicular fluid was collected by aspiration from 3 to 6 mm follicles with an 18-gauge needle and allowed sediment to settle to the bottom of 50 mL conical tubes held at 37℃. The sediment was pooled and washed three times in washing medium comprising 9.5 g/L of tissue culture medium-199 (TCM-199; Invitrogen), 5 mM sodium hydroxide, 2 mM sodium bicarbonate, 10 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 0.3% polyvinyl alcohol, and 1% penicillin-streptomycin (Invitrogen). Only cumulus-oocyte complexes (COCs) with homogeneous cytoplasm and three or more layers of cumulus cells were collected and placed into in vitro maturation (IVM) medium containing TCM-199 supplemented with 2 mM sodium pyruvate, 5 µL/mL insulin transferrin selenium solution 100X (Invitrogen), 0.57 mM cysteine, 10 ng/mL epidermal growth factor, 10% porcine follicular fluid (v/v), 10 IU/mL human chorionic gonadotropin, and 10 IU/mL equine chorionic gonadotropin at 38.5℃ under 5% CO2 in 95% humidified air. After 22 h of IVM, the COCs were washed and culture was continued in hormone-free IVM medium for an additional 22 h.
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