Preparation of rat serum suitable for mammalian whole embryo culture.
Preparation of rat serum suitable for mammalian whole embryo culture.
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DOI：
10.3791/51969
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发表时间：
2014-08
期刊：
影响因子：
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通讯作者：
Masanori Takahashi;Sayaka Makino;Takako Kikkawa;N. Osumi
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文献类型：
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作者：
Masanori Takahashi;Sayaka Makino;Takako Kikkawa;N. Osumi
哺乳动物全胚胎培养（WEC）是一种广泛用于检查小鼠和大鼠胚胎发育中的药理学毒性以及研究发育过程机制的技术。立即离心（IC）大鼠血清通常用于WEC，并且对于体外培养的小鼠和大鼠胚胎的生长和发育至关重要。对于妊娠中期胚胎的培养（即，小鼠E8.0-12.5，大鼠E10.0-14.5），100%大鼠血清是支持胚胎离体生长的最佳培养基。为制备适用于WEC的大鼠血清，应立即离心采集的血液，以将血细胞与血浆组分分离。离心后，上层形成纤维蛋白凝块;应使用一对无菌镊子轻轻挤压凝块，随后离心，以完全分离血细胞和血清。在这篇视频文章中，我们展示了我们制备最佳IC大鼠血清的标准方案，包括从雄性大鼠腹主动脉采血和离心提取血清。
Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.