To quantitatively analyze apoptotic and secondary necrotic cells unde r apoptosis conditions. Methods. The cells of Burkitt lymphoma (BL) cell line Raji were incubated with 1 .0 μmol/L dexamethasone (DEX) for 2, 4 an...To quantitatively analyze apoptotic and secondary necrotic cells unde r apoptosis conditions. Methods. The cells of Burkitt lymphoma (BL) cell line Raji were incubated with 1 .0 μmol/L dexamethasone (DEX) for 2, 4 and 8 h respectively, then stained with Annexin V FITC (fluorescein isothiocyanate conjugated) which was used to detec t the exposed phosphatidylserine (PS) on the epimembrane resulting from a loss o f phospholipid asymmetry in the early stage of apoptosis, and also stained with propidium iodide (PI) which allows analysis of secondary necrotic cells related with cell membrane and DNA damage that probably represent late stage of apoptosi s, then apoptotic cells were quantified by flow cytometry (FCM). Furthermore, An nexin+/PI and Annexin+/PI+ cells were sorted by fluoresence activated cell sorter (FACS), and identified by electron microscopy (EM) and DNA gel electroph oresis. Results. The percentage of apoptotic cells was found to increase with the incuba tion time (r=0.97). This method was sensitive with low detection limit (0.02%), and was reproducible with low coefficient variance (CV)(4.2%). Meanwhile, the Annexin+/PI and Annexin+/PI+ cells were identified as apoptotic and necroti c cells under EM, and DNA extracted from the Annexin+/PI cells was characteri zed by “ladder pattern”. Conclusions. Annexin V assay is a specific, sensitive, accurate, reproductive and quantitative method for analyzing apoptotic cells.展开更多
目的制备人膜联蛋白 V(Annexin V),应用联肼尼克酰胺(HYNIC)偶联后进行^(99)Tc^m 标记,评价^(99)Tc^m-HYNIC-Annexin V 在健康小鼠体内的分布。方法采用基因工程方法构建 Annexin V 的原核载体并诱导其表达,与自行合成的双功能螯合剂 HY...目的制备人膜联蛋白 V(Annexin V),应用联肼尼克酰胺(HYNIC)偶联后进行^(99)Tc^m 标记,评价^(99)Tc^m-HYNIC-Annexin V 在健康小鼠体内的分布。方法采用基因工程方法构建 Annexin V 的原核载体并诱导其表达,与自行合成的双功能螯合剂 HYNIC 偶联,并进行^(99)Tc^m 标记。测定^(99)Tc^m-HYNIC-Annexin V 的标记率、放化纯,并研究其在健康小鼠体内的生物分布特性。结果 Annexin V在大肠杆菌中获得稳定表达,经十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)及 Western Blot-ting 检测,得到纯度较高的蛋白质。Annexin V 经 HYNIC 偶联后,其^(99)Tc^m 标记率可达90%左右,放化纯>90%。小鼠体内分布结果表明,^(99)Tc^m-HYNIC-Annexin V 血液清除迅速,主要从肾脏和肝脏排泄。结论 Annexin V 可在原核生物中稳定表达。其与 HYNIC 偶联后,^(99)Tc^m 标记率和放化纯较高,方法简单,条件温和。展开更多
文摘To quantitatively analyze apoptotic and secondary necrotic cells unde r apoptosis conditions. Methods. The cells of Burkitt lymphoma (BL) cell line Raji were incubated with 1 .0 μmol/L dexamethasone (DEX) for 2, 4 and 8 h respectively, then stained with Annexin V FITC (fluorescein isothiocyanate conjugated) which was used to detec t the exposed phosphatidylserine (PS) on the epimembrane resulting from a loss o f phospholipid asymmetry in the early stage of apoptosis, and also stained with propidium iodide (PI) which allows analysis of secondary necrotic cells related with cell membrane and DNA damage that probably represent late stage of apoptosi s, then apoptotic cells were quantified by flow cytometry (FCM). Furthermore, An nexin+/PI and Annexin+/PI+ cells were sorted by fluoresence activated cell sorter (FACS), and identified by electron microscopy (EM) and DNA gel electroph oresis. Results. The percentage of apoptotic cells was found to increase with the incuba tion time (r=0.97). This method was sensitive with low detection limit (0.02%), and was reproducible with low coefficient variance (CV)(4.2%). Meanwhile, the Annexin+/PI and Annexin+/PI+ cells were identified as apoptotic and necroti c cells under EM, and DNA extracted from the Annexin+/PI cells was characteri zed by “ladder pattern”. Conclusions. Annexin V assay is a specific, sensitive, accurate, reproductive and quantitative method for analyzing apoptotic cells.
文摘目的制备人膜联蛋白 V(Annexin V),应用联肼尼克酰胺(HYNIC)偶联后进行^(99)Tc^m 标记,评价^(99)Tc^m-HYNIC-Annexin V 在健康小鼠体内的分布。方法采用基因工程方法构建 Annexin V 的原核载体并诱导其表达,与自行合成的双功能螯合剂 HYNIC 偶联,并进行^(99)Tc^m 标记。测定^(99)Tc^m-HYNIC-Annexin V 的标记率、放化纯,并研究其在健康小鼠体内的生物分布特性。结果 Annexin V在大肠杆菌中获得稳定表达,经十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)及 Western Blot-ting 检测,得到纯度较高的蛋白质。Annexin V 经 HYNIC 偶联后,其^(99)Tc^m 标记率可达90%左右,放化纯>90%。小鼠体内分布结果表明,^(99)Tc^m-HYNIC-Annexin V 血液清除迅速,主要从肾脏和肝脏排泄。结论 Annexin V 可在原核生物中稳定表达。其与 HYNIC 偶联后,^(99)Tc^m 标记率和放化纯较高,方法简单,条件温和。
