ANNEXIN V- FITC/PI 凋亡检测试剂盒.








产品说明：
细胞凋亡是细胞的基本特征之一，在机体的胚胎发育、组织修复、内环境的稳定等方面都起到 十分重要的作用。Annexin V 是一种分子量为 35.8 KD 的 Ca2+依赖性磷脂结合蛋白，能与细胞凋亡 过程中翻转到膜外的磷脂酰丝氨酸（Phosphatidylserine, PS）高亲和力特异结合。FITC-Annexin V 结 合到凋亡细胞后，在蓝色光的激发下，发出绿色荧光，区分出凋亡细胞与正常细胞。碘化丙啶 （Propidium iodide，PI）是一种核酸染料，它不能穿透完整细胞膜，但对凋亡晚期细胞和死细胞的 破损细胞膜能够穿透，并使细胞核红染。将 FITC-Annexin V 与PI 匹配使用，可以将凋亡早期的细 胞和晚期的细胞区分开来。 :

操作步骤
1、 用 27ml 的去离子水稀释 3ml Binding Buffer（10×）至 30ml，每次用 3ml。
2、 收集细胞（1×106 个/次），然后用冷的 PBS 洗涤。（对于贴壁细胞，先用胰酶消化，再用 PBS 洗 涤）。
3、 用 1ml 1×的 Binding Buffer 悬浮细胞，300×g 离心 10min，弃上清。
4、 用 1ml 1×的 Binding Buffer 重悬细胞，使细胞的密度达到 1×106 个/ml。
5、 每管加入 100μL 细胞（1×105 个）。
6、 再向管中加入 5μL Annexin V-FITC。
7、 室温，避光，轻轻地混匀，10min。
8、 加入 5μL PI，室温，避光，孵育 5min。
9、 加入 PBS 至 500μL，轻轻混匀。
10、在 1 小时内用流式细胞仪检测。

注意事项
1、旋帽离心管装的试剂在开盖前请短暂离心，将盖内壁上的液体甩至管底，避免开盖时液体洒落。
2、Annexin V-FITC 和 Propidium iodide 是光敏物质，在保存与操作时请注意避光。
3、Propidium iodide（PI）能通过皮肤吸收，对眼睛有刺激作用。
4、在细胞洗涤的*一步，请尽量将上清弃净，以免 PBS 残留影响实验结果。 产品内容: CA1020-20 CA1020-50 CA1020-100 保存 Annexin V-FITC 100μl 250μl 500μl 4°C，避光，不要冷冻 Propidium iodide(PI) 100μl 250μl 500μl 4°C，避光，不要冷冻 Binding Buffer（10×） 6ml 15ml 30ml 4°C 保存，长时间-20°C
5、PI 染色时间过长有可能造成检测的凋亡率偏高，建议首先进行 Annexin V-FITC 染色，上机前 5 分钟再加入PI 染色。
6、整个操作过程动作要尽量轻柔，勿用力吹打细胞，尽量在 4℃下操作。
7、反应完毕后请尽快检测，因为细胞凋亡是一个动态的过程，反应 1 小时后荧光强度就开始衰变。
8、成功的检测凋亡受以下几种因素的影响，如细胞类型、细胞膜上 PS 的密度、发生凋亡时 PS 翻 转的比例、诱导细胞凋亡的方法、所用试剂、诱导凋亡的时间等，把这些影响因素进行优化对实验 成功是非常必要的。 此试剂盒仅供科研使用。


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CA1030   Annexin V PE/7-AAD 凋亡检测试剂盒

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《Mechanism of miR-15a regulating the growth and apoptosis of human knee joint chondrocytes by targeting SMAD2》 作者:Tengjun Ma, Yan Cheng & Liang Tan 期刊:Artif Cells Nanomed Biotechnol 影响因子:4.462 PMID:31366242

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