AT101C

Annexin V-FITC/PI Apoptosis Kit(贴壁细胞专用)(C6流式细胞仪专用)

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¥860.00¥1,510.00

在售SKU:70-AT101C-100, 70-AT101C-60, 70-AT101C-30, 凋亡, 细胞凋亡, 凋亡试剂盒, 细胞凋亡试剂盒, 凋亡检测, 细胞凋亡检测, 凋亡检测试剂盒, 细胞凋亡检测试剂盒, Annexin V, Annexin, Apoptosis, 贴壁, 贴壁细胞, 贴壁细胞专用, C6

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描述

背景简介

Annexin V(或 Annexin A5)为胞内蛋白膜联蛋白家族成员,以钙依赖的方式与磷脂酰丝氨酸(PS)结合。PS 存在于正常细胞浆膜的内层,但在凋亡早期,膜不对称性丧失,PS 易位至细胞表面。荧光标记的 Annexin V 可与之特异性结合,表明该细胞为凋亡细胞。

PI(碘化丙啶)是一种核酸染料,它不能透过正常细胞或早期凋亡细胞的完整的细胞膜,但可以透过凋亡晚期和坏死细胞的细胞膜而使细胞核染红。因此,将 Annexin V 与PI 联合使用时,PI 则被排除在活细胞(Annexin V- / PI-)和早期凋亡细胞(Annexin V+ / PI-)之外,而晚期凋亡细胞和坏死细胞同时被荧光标记的Annexin V 和 PI 结合染色呈现双阳性(Annexin V+ / PI+)。

Accutase 是一种天然的酶,兼具蛋白水解酶和胶原酶的活性。但它比胰酶和胶原酶更有效,使用浓度更低,使之毒性更小、更温和。因此对细胞的损伤更小,可减少因消化处理带来的假阳性。即便长时间消化, 也可保持90%以上的细胞活力。适合于各种贴壁细胞、胚胎和神经元干细胞等较敏感的细胞的消化。

本试剂盒仅需 15 分钟即可染色样本,快速方便。提供阳性质控液,便于制备单染管,调节补偿。提供消化细胞更温和的 Accutase,减少假阳性。

注:此凋亡试剂盒针对BD的C6流式细胞仪器进行优化。

试剂盒组分

目录号 AT101C-30 AT101C-60 AT101C-100 储存条件
规格 30T 60T 100T
Annexin V-FITC 150 μl 300 μl 500 μl 2 - 8℃,避光
PI 300 μl 600 μl 1000 μl 2 - 8℃,避光
5 × Binding Buffer 5 ml 10 ml 15 ml 2 - 8℃
Apoptosis Positive Control Solution 5 ml 5 ml 5 ml 2 - 8℃
Accutase 100 ml 100 ml 100 ml -20℃,解冻后-20℃分装保存,或2 - 8℃可保存2 个月

使用方法

一、 细胞消化(不同类型的细胞,需要用户优化方案)

  1. 将 Accutase 于4℃或室温解冻,切勿放于 37℃解冻。
  2. 小心吸去细胞培养基 (如培养上清中存在凋亡细胞,则应收集)。
  3. (可选) 无菌PBS 洗涤。
  4. 加入适量解冻的 Accutase (不必温热至 37℃),覆盖住细胞。根据细胞汇合度和密度情况,通常在T25细胞瓶中加入 2.5 - 5 ml Accutase。
  5. 室温放置 5 - 10 分钟,至多可长达 1 小时,无需终止。
  6. 当细胞开始圆缩,用手掌轻拍培养瓶,使细胞脱落。
  7. 温和地吹散细胞,收集细胞。

仪器参数调节

  1. 收集1 × 106- 3 × 106 个细胞,用预冷 PBS 离心洗涤两次,弃上清。
  2. 加入500 μl Apoptosis Positive Control Solution 重悬,置冰上孵育 30 分钟。
  3. 用预冷PBS 离心洗涤,弃上清。
  4. 加入适量预冷 1 × Binding Buffer 重悬,并加入数量相同且未经处理的活细胞与之混合。加入预冷1×Binding Buffer 补充至1.5 ml,等分成三管,其中一管为空白对照管、两管为单染管。5.单染管分别加入 5 μl Annexin V-FITC 或10 μl PI,室温避光孵育 5 分钟。6. 在流式细胞仪上,用空白管调节 FSC、SSC 和荧光通道的电压,并在此电压条件下,用单染管调节荧光通道的补偿。

三、 样本检测

  1. 按实验方案诱导凋亡。
  2. 用预冷 PBS 离心洗涤,收集1 - 10 × 105 个细胞(包括培养上清中的细胞)。用双蒸水稀释5 × BindingBuffer 为 1 × 工作液,取 500 μl 1 × Binding Buffer 重悬细胞。
  3. 每管加入5 μl Annexin V-FITC 和10 μl PI。
  4. 轻柔涡旋混匀后,室温避光孵育 5 分钟。
  5. 根据实验方法,进行流式分析。
  6. 流式分析
    在流式细胞仪上,通过FITC 检测通道检测 Annexin V-FITC (Ex = 488 nm; Em = 530 nm)和通过PI 检测通道(Ex = 535 nm; Em = 615 nm)检测PI。

结果示例

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