Ovaries were excised and transferred into M2 medium (Merck) containing 0.1 mM 3-isobutyl-1-methylxanthine (IBMX, Merck) to keep immature oocytes. Germinal vesicle (GV) stage oocytes were isolated and subsequently cultured at 37 °C in 5% CO2 in M16 medium (Merck) with IBMX, covered with mineral oil (Merck or YBUX) for at least 1 hour prior to microinjection. Zygotes and 2-cell embryos were isolated from oviducts 18–21 and 45–47 hours post hCG stimulation in M2 medium (Merck) and subsequently cultured in KSOM + AA (Caisson Laboratories) covered with mineral oil (Merck or YBUX) at 37 °C, 5% CO2. For removing of the cumulus cells was used 0.05% hyaluronidase from bovine testes (Merck). Microinjection was performed in M2 medium with (oocytes) or without (embryos) IBMX inhibitor using I10 Narishige microinjector on a Leica DM IL inverted microscope. After microinjection were oocytes (M16 medium + IBMX, Merck) and embryos (KSOM + AA, Caisson Laboratories) cultured for cRNA expression for 1 to 3 hours prior to live cell imaging assay (37 °C, 5% CO2).
Ksom aa
Manufactured by Caisson
KSOM + AA is a type of cell culture media used for the in vitro cultivation of embryos. It provides the necessary nutrients and growth factors required for embryo development.
Automatically generated - may contain errors
2 protocols using ksom aa
1
Murine Oocyte and Embryo Microinjection
2
Isolation and Culture of Mouse Morula Embryos
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Embryos were isolated from superovulated female ICR mice mated with male mice. Superovulation of female mice is induced by intraperitoneal injection of 10 international units (IU) of pregnant mare's serum gonadotropin (PMSG), followed by intraperitoneal injection of 10 IU human chorionic gon adotropin (hCG) 44-48 hr later. Morula-stage (E2.5) embryos were recovered by flushing uterus from plugged females with 37 C M2 medium (Sigma) using an injection syringe (zymm HX-Z08). Embryos were handled using an aspirator tube equipped with a glass pipette pulled from glass micropipettes. Embryos were placed in KSOM+AA (caisson, IVL04) in 20-ml droplets covered in mineral oil (Sigma). Embryos were cultured in an incubator with a humidified atmosphere supplemented with 5% CO 2 at 37 C. Experiments were replicated at least three times.
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