流式细胞术Caspases酶检测

细胞凋亡早期阶段的显著特征是caspase酶的活化,其参与了蛋白底物剪切以及随后的细胞裂解。 Life Technologies™ provides a series of caspase assays that allow the simple detection of active caspases in living cells by flow cytometry.

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快速链接

CellEvent® Caspase-3/7 Green Flow Cytometry Assay Kit

The CellEvent® Caspase-3/7 Green Flow Cytometry Assay Kit enables the flow cytometric detection of activated caspase-3 and caspase-7 in apoptotic cells. The kit includes the novel fluorogenic substrate CellEvent® Caspase-3/7 Green Detection Reagent, as well as SYTOX® AADvanced™ Dead Cell Stain.

  • Caspase-3/7 specific—includes the recognition sequence for activated caspase-3 and caspase-7
  • Easy identification—clearly identify live, dead, and apoptotic cell populations
  • Quick analysis—washing and fixation is not required
  • Multicolor compatibility—combine with other dyes excitable by the 488 nm laser or other lasers

CellEvent® Caspase-3/7 Green Detection Reagent is a cell-permeant reagent that consists of a four-amino acid peptide (DEVD) conjugated to a nucleic acid-binding dye. During apoptosis, caspase-3 and caspase-7 proteins are activated and able to cleave the caspase 3/7 recognition sequence encoded in the DEVD peptide. Cleavage of the recognition sequence and binding of DNA by the reagent labels the apoptotic cells with a bright, fluorogenic signal that has absorption/emission maxima of ~511/533 nm. When used together with the SYTOX® AADvanced™ Dead Cell Stain, apoptotic cells can be easiliy discriminated from live and necrotic cells.

 

 

CellEvent® Caspase-3/7 Green Flow Cytometry Assay Kit

 
Caspase activity detection in Jurkat cells using the CellEvent® Caspase-3/7 Green Flow Cytometry Assay Kit on the Attune® Flow Cytometer. Jurkat cells (T-cell leukemia, human) were treated with (A) DMSO or (B) 10 µM camptothecin for 3 hours before labeling with the CellEvent® Caspase 3/7 Green Flow Cytometry kit. Stained samples were analyzed on the Attune® Acoustic Focusing Cytometer equipped with a 488-nm laser, and fluorescence emission was collected using a 530/30 BP filter for CellEvent® Caspase 3/7 Green Detection Reagent and a 690/50BP filter for SYTOX® AADvanced™ stain, respectively. Note that the treated cells have a higher percentage of apoptotic cells (panel B) than the basal level of apoptosis seen in the control cells (panel A). A =apoptotic cells, V = viable cells, N = necrotic cells.

 

产品 激光 激发/发射光波长 目录号  
CellEvent® Caspase-3/7 Green Flow Cytometry Assay Kit 488 511/533 C10427 产品详情

Vybrant ® FAM™ Caspase Assay Kits

Vybrant® FAM™ Caspase分析试剂盒采用基于caspase荧光抑制剂 (FLICA®) 的新方法,检测活细胞中活化的caspase。 Vybrant® FAM™ Caspase检测试剂盒使用了一种新型的caspase抑制剂——FLICA®试剂,该试剂可与活化的caspase的活性半胱氨酸发生不可逆的共价结合,抑制进一步的酶活性 (图1)。

该方法使用简单,不需要特殊的缓冲液裂解或通透细胞膜——只需添加试剂至您的细胞培养基中,孵育1–4个小时后,清洗细胞并分析即可。

  • 可在活细胞中准确检测Caspases酶活性
  • 比膜联蛋白V更可靠
  • 比TUNEL分析更简单

 


图1. 利用Vybrant® FAM™ Caspase分析试剂盒检测凋亡细胞中活化的caspase。 未结合caspase的FLICA®试剂会扩散到细胞外,被清洗掉。 绿色荧光信号直接代表了加入抑制剂时活化caspase的含量。

  图2. 流式细胞术检测活细胞内活化的Caspase。 Jurkat细胞用10 μM喜树碱处理 (37ºC、5% CO2下温育4小时) 后,用检测Caspase-3酶和-7的FLICA®试剂以及碘化丙啶染色,两者均来自于Vybrant® FAM™ Caspase-3和-7分析试剂盒。在流式细胞仪上使用530 nm带通和670 nm长通发射滤光片,以488 nm激发光分析样品。

 

偶联物 检测到的Caspase
激光 激发/发射光波长 目录号  
Vybrant® FAM™ Poly caspase分析试剂盒 全部 488 495/529 V35117 产品详情
Vybrant® FAM™ caspase-3 & -7分析试剂盒 3,7 488 495/529 V35118 产品详情
Vybrant® FAM™ caspase-8分析试剂盒 8 488 495/529 V35119 产品详情

PARP蛋白水解

凋亡过程中,ICE家族成员如caspase-3和-7可分解PARP,产生85 kDa和25 kDa的片段。 PARP剪切被认为是凋亡过程的典型特征之一。 FITC偶联的抗PARP抗体特异性识别PARP水解的85 kDa片段,其可用作检测凋亡细胞的标志物。 抗PARP FITC细胞凋亡试剂盒包含所有试剂以及使用流式细胞仪分析PARP裂解所需的简要的实验方案。


图3. 使用FITC-PARP剪切位点特异性抗体,流式细胞仪检测凋亡的Hela细胞 (贴壁培养)。 Hela细胞采用0.5 μM星形孢菌素37ºC处理5小时 (下图),未诱导的Hela细胞作为对照组 (上图)。 细胞用10 μL FITC偶联的抗PARP CSSA染色30分钟,然后进行FACScan分析。 数据显示,星形孢菌素处理后发生凋亡的Hela细胞可被PARP CSSA染色 (左下),而未诱导Hela细胞未被染色 (左上)。 凋亡细胞的该阳性染色可被人PARP N-端剪切位点214/215相应的合成肽所封闭,这表明FITC-PARP CSSA对凋亡细胞的染色是特异的 (右下)。

产品 激光 激发/发射光波长 目录号  
抗PARP FITC凋亡试剂盒 488 495/529 AHM2011 产品详情

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