培养小鼠逆转录病毒包装株 PT-67细胞操作
1. 器械和器皿
器械:外科剪、镊子、虹膜剪等、小剪刀、细镊子和虹膜小刀等
各种金属器械如解剖器械,使用后及时刷洗干净,用酒精棉球擦拭后晾干,或经60°C烘干,以防生锈。由于湿热消毒对手术器械容易可用70-80%酒精浸泡1小时以上消毒。
器皿:1) 玻璃瓶及相应的胶塞或胶木螺旋盖
用于分装血清、多聚赖氨酸、解剖液、小鼠逆转录病毒包装株 PT-67细胞各种盐溶液和培养液等。
2)用培养瓶、盖玻片
培养用的盖玻片必须不含铅、不发霉,可用0.2N盐酸浸泡10分钟,小鼠逆转录病毒包装株 PT-67细胞用蒸馏水洗2次,每次10分钟,然后移入丙酮或乙醇浸泡10分钟,再用双蒸水洗2次,每次10分钟,烘干待消毒。凡组织学用过的旧盖玻片一般不用。
3) 移液管、吸管、烧杯、离心管和培养皿。
4) 塑料培养皿(35mm)、塑料培养板(6、24、96孔)。
辅助工具:放置试管、吸管和玻璃瓶等的架子。
细胞识别,根据细胞外形及内部结构进行细胞的识别。神经细胞具有显著的特征,培养中的贴壁的神经元突起很明显,特别是树突由粗而细,有分支,突起的末端有膨大的生长锥结构,正在不断变化;细胞体呈圆形、梭形、三角形或多角形不等,小鼠逆转录病毒包装株 PT-67细胞胞体较厚,具有空泡状核,有明显的核仁;立体感强,常见“晕”。
培养细胞中可包括几种神经元的类型和非神经元的“背景细胞”,即成纤维细胞、室管膜细胞和几种胶质细胞。成纤维细胞贴壁最快,呈扁平状,胞核卵园形,有并列的两粒小核仁,从胞体两端发出细长突起。胶质细胞贴附在胶原上和成纤维细胞形成一层“地毯”。神经细胞贴壁较慢,落在“地毯”上生长迁移和分化。
培养的神经细胞可按一般Nissl法染色或免疫细胞化学特异性染色加以鉴别。
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