The zygotes and oocytes were recovered from the excised oviducts (15–18 hr after the hCG injection) by dissection of oviduct wall into M2 medium (Sigma, USA) containing 0.1% (w/v) hyaluronidase (Sigma, USA) to remove cumulus cells. In vivo produced 2-cell embryos were recovered from the excised oviducts (45–48 hours after hCG) by M2 medium flushing via oviduct ampulla using fine capillary connected with syringe. Then the ova were washed in M2 medium and used for manipulations. For in vitro cultivation, 2-cell embryos were transferred to four-well culture dishes (Nunc, Denmark) and cultured in M16 medium (Sigma, USA) under 5% CO2 in air at 37 °C. Previously, the culture medium was equilibrated with the gas phase and temperature in a CO2 incubator for 2–3 hours. The same method of cultivation was used for estimation of embryo viability after laser treatment.
Four well culture dishes
Manufactured by Thermo Fisher Scientific
Sourced in Denmark
Four-well culture dishes are multi-well cell culture plates designed for the in vitro cultivation and study of cells. Each dish contains four individual wells for culturing and experimenting with different cell lines or experimental conditions simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
Hyaluronidase, by Merck Group (1 mentions)
M2 medium, by Merck Group (1 mentions)
M16 medium, by Merck Group (1 mentions)
Fluoxetine hydrochloride, by Merck Group (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Serotonin creatinine sulfate, by Merck Group (1 mentions)
H9772, by Merck Group (1 mentions)
5 hydroxy l tryptophan, by Merck Group (1 mentions)
Tcm 199, by Thermo Fisher Scientific (1 mentions)
4 protocols using four well culture dishes
1
Isolation and Culture of Mouse Embryos
2
Ovarian Culture with Serotonergic Agents
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Mouse ovaries at 14 dpp were isolated, dissected into eight fragments and cultured in four-well culture dishes (Nunc, Roskilde, Denmark) containing 1 mL DMEM/F-12 medium (Gibco, Gaithersburg, MD, USA) supplemented with chemicals at 37 °C in 5% CO2. The chemicals used in the study were serotonin creatinine sulfate (1 μM; H7752 Sigma-Aldrich, St. Louis, MO, USA), fluoxetine hydrochloride (10 μM; PHR1394 Sigma-Aldrich, St. Louis, MO, USA) and 5-hydroxy-L-tryptophan (10 μM; H9772 Sigma-Aldrich, St. Louis, MO, USA). The ovaries from three to five animals were used for each experiment.
3
Thawing and Culturing Frozen Cells
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For thawing cells, five days prior to use, frozen cells were warmed in 37°C sterile water, and the culture media was added to the cells. To remove DMSO, the cells were centrifuged at 400 g for 5 min and re-suspended in warm culture media and cultured into four-well culture dishes (Nunc , Denmark) with a concentration of 1 10 cell/ml. During the cell culture, the media was refreshed every 48 hr. One day before co-culturing the oocytes with BAOECs, the culture media was substituted by maturation media (14).
4
Porcine Oocyte Maturation in vitro
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Ovaries were collected from 5- to 6-month-old prepubertal gilts (100 ± 10 kg body weight) from a local slaughterhouse, placed in saline at 30~35℃, and transported to the laboratory within 2 h. After washing with saline three times, cumulus-oocyte complexes (COCs) were recovered by aspiration of 2- to 5-mm follicles using an 18-gauge hypodermic needle attached to a 5 mL disposable syringe (Korea Vaccine, Korea). After washing three times in IVM medium, COCs that were enclosed by more than three layers of compact cumulus cells and an evenly granulated ooplasm were selected for in vitro maturation. The 200~250 selected COCs per were cultured in four-well culture dishes (Nunc, Denmark) containing 500 µL of IVM medium under warmed and gas-equilibrated mineral oil for 44 to 46 h at 38.5℃ and 5% CO2. The IVM medium for oocytes was composed of tissue culture medium 199 with Earle's salts and L-glutamine (TCM199; Life technologies) supplemented with 26.2 mM NaHCO3, 3.05 mM glucose, 0.91 mM sodium pyruvate, 0.57 mM L-cysteine, 10 ng/mL epidermal growth factor (EGF), 1 µg/mL insulin, 10 IU/mL equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG), and 0.1% (w/v) polyvinyl alcohol (PVA) [3 (link)].
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