Annexin V-FITC/PI细胞凋亡检测试剂盒

- Annexin V-FITC apoptosis assay kit
货号-规格 | 货期 | 价格 | 数量 |
abs50001-25T | 现货 | ¥630.00 | - + |
abs50001-50T | 1-2周 | ¥1050.00 | - + |
abs50001-100T | 现货 | ¥1838.00 | - + |

产品描述 | |||||||||||||||||
描述 | 在正常细胞中,磷脂酰丝氨酸只分布在细胞膜脂质双层的内侧,细胞发生凋亡最早期,膜磷脂酰丝氨酸(PS)由脂膜内侧翻向外侧,这一变化早于细胞皱缩、染色质浓缩、DNA片断化和细胞膜的通透性增加等凋亡现象。Annexin V是一种磷脂结合蛋白,与磷脂酰丝氨酸有高度亲和力,故可通过细胞外侧暴露的磷脂酰丝氨酸与凋亡早期细胞的胞膜结合。因此Annexin V被作为检测细胞早期凋亡的灵敏指标之一。碘化丙啶(Propidium Iodide, PI)是一种核酸染料, 它不能透过完整的细胞膜,但凋亡中晚期的细胞和死细胞由于细胞膜通透性的增加,PI 能够透过细胞膜而使细胞核染红。因此将Annexin V 与PI 匹配使用,就可以将处于不同凋亡时期的细胞区分开来。 | ||||||||||||||||
产品组分 |
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操作步骤 | 贴壁细胞需用 0.25%的胰酶消化。注意过度消化可损伤细胞。在消化时可加 2%的BSA可防止消化过度。如果用含EDTA的胰酶消化时,注意必须彻底清除EDTA:在标记前用 1×PBS或 1×binding buffer洗涤,清除EDTA,以免残余的EDTA与Ca2+螯合,影响Annexin V的结合。 (1) 用去离子水将 10×Binding Buffer 稀释成 1×Binding Buffer; (2) 细胞收集:悬浮细胞收集:离心 5 分钟;贴壁细胞:用不含EDTA的胰酶消化收集后( 注:胰酶消化时间不宜过长,否则会影响细胞膜上磷脂酰丝氨酸与Annexin V-FITC 的结合),在室温中,2000rpm 离心 5~10 分钟,收集细胞; (3) 细胞洗涤:用预冷 1×PBS(4℃)重悬细胞一次,2000rpm 离心 5~10分钟,弃上清洗涤细胞; (4) 加入 300μL 的 1×Binding Buffer 悬浮细胞; (5) Annexin V-FITC 标记:加入 5μL 的 Annexin V-FITC 混匀后,避光,室温孵育 15 分钟; (6) PI 标记:上机前 5 分钟再加入 5μL 的 PI 染色。 (7) 上机前,补加 200μL 的 1×Binding Buffer。 注意:每个反应体系建议细胞数为1×104至1×106个 | ||||||||||||||||
操作说明 | (1) 使用前低速离心,以免液体积存管盖和管壁;
(2) 本试剂只适用于实验,不适用于临床检测; (3) PI(propidium iodide,溴化丙锭)有毒性,能通过皮肤吸收,对眼睛有刺激作用,使用时需戴手套; (4) Annexin V-FITC 和 PI 是光敏物质,在操作时请注意避光。在处理和标记时,尽可能在暗处进行。在孵育阶段,用铝箔包裹容器或置于抽屉中。细胞标记后,在暗室内用显微镜观察; (5) 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,以免影响细胞状态。 (6) 在细胞洗涤的最后一步,请尽量将上清弃净,以免 PBS 残留,有可能会影响实验结果。 (7) 为防止荧光衰变,宜在 1 小时内进行流式检测。 (8) 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,最短可在上机前 5 分钟再加入 PI 染色。 | ||||||||||||||||
基本信息 | |||||||||||||||||
别名 | Annexin V-FITC apoptosis assay kit | ||||||||||||||||
储存/保存方法 | 4℃保存6个月;避免反复冻融。 | ||||||||||||||||
温馨提示:本产品仅作科研实验使用,不支持临床等研究 |
- 实验方法 实验条件
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- 有哪些注意事项
- 1. 由于凋亡检测的为活细胞,因此整个收集细胞的操作过程尽量保持细胞活性,避免人为损伤细胞, 如:避免用力吹打细胞,避免胰酶消化时间过长,用冰浴的PBS清洗细胞,缩短上机检测的时间。 2. Annexin-V染料上带FITC荧光,因此从染色开始注意避光操作和孵育,一般尽量在染色后半个小时内 上机检测。 3. 如果处理的细胞中含有GFP会干扰FITC的荧光信号,应该更换不同波长的荧光染料标记的Annexin-V。 4. 如果样品来源于血液,请务必除去血液中的血小板,因为血小板含有PS,能与Annexin-V结合, 从而干扰实验结果。 5. 在消化液中尽量不用EDTA,EDTA会影响Annexin-V与PS的结合,若胞内已转染GFP, 建议使用AnnexinV-PE。 6. 如果样本来源为贴壁细胞,建议使用Accutase酶。 7. 建议细胞密度为1-5*106/ml,为保证实验结果,最佳细胞密度需自行摸索。
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