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Cell Meter 通用Caspase活性荧光法检测试剂盒 绿色荧光 适合于流式细胞检测

英文名称:Cell Meter™ Generic Fluorimetric Caspase Activity Assay Kit *Green Fluorescence Optimized for Flow Cytometry*
产品参数
Ex (nm)503Em (nm)525
分子量-溶剂-
存储条件-
产品概述

我们的Cell Meter 检测试剂盒是一套用于检测细胞生存力的工具。有多种参数可用于检测细胞活力。该特定试剂盒旨在通过测量活细胞中的通用胱天蛋白酶(胱天蛋白酶-1,-3,-4,-5,-6,-7,-8和-9)来检测细胞凋亡。胱天蛋白酶被广泛认为是细胞凋亡的可靠指标。大多数胱天蛋白酶具有对肽序列Val-Ala-Asp(VAD)的底物选择性。该试剂盒使用TF2-VAD-FMK作为大多数半胱天冬酶活性的荧光指示剂。TF2-VAD-FMK是细胞渗透性的,并且与凋亡细胞中活化的casepase-1,-3,-4,-5,-6,-7,-8和-9不可逆地结合。一旦与胱天蛋白酶结合,绿色荧光试剂将保留在细胞内。结合后阻止了胱天蛋白酶的进一步催化,但不会阻止凋亡的进行。添加到培养基中后的15分钟内,该试剂将开始与活性caspase酶反应。该试剂盒为所有必需成分提供了优化的测定方案。它用于定量凋亡细胞中大多数胱天蛋白酶活性,或筛选胱天蛋白酶抑制剂。绿色荧光标记允许通过流式细胞仪直接检测凋亡细胞胱天蛋白酶。百萤生物是AAT Bioquest的中国代理商,为您提供优质的Cell Meter 通用Caspase活性荧光法检测试剂盒

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适用仪器


流式细胞仪  
Ex: 488 nm
Em: 530/30 nm
通道: FITC 通道
实验方案

样品实验方案

简要概述

  1. 用5×105至1×106细胞/ mL的密度制备含测试化合物的细胞
  2. 将0.5 µL细胞溶液中加入1 µL 500X TF2-VAD-FMK
  3. 将细胞在37°C,5%CO2培养箱中孵育1-4小时
  4. 沉淀细胞并将细胞重悬于0.5 mL检测缓冲液或生长培养基中
  5. 使用带有530/30 nm滤光片(FITC通道)的流式细胞仪分析细胞

 

实验步骤

1.对于每个样品,在0.5 mL温热培养基或自备缓冲液中以5×105至1×106cell/ mL的密度制备细胞。注意:应单独评估每种细胞系,以确定诱导凋亡的佳细胞密度。

2.用测试化合物处理细胞一段时间,以诱导细胞凋亡,并建立阳性和阴性对照。

3.向处理过的细胞中加入1µL 500X TF2-VAD-FMK(组分A)。

4.将细胞在37°C,5%CO2培养箱中孵育1-4小时。注意:对于贴壁细胞,用0.5 mM EDTA轻轻提起细胞以保持细胞完整,并在与TF2-VAD-FMK孵育之前用含血清的培养基洗涤细胞一次。合适的孵育时间取决于所用的单个细胞类型和细胞浓度。优化每个实验的孵育时间。

5.洗涤并旋转细胞两次。将细胞重悬于0.5 mL分析缓冲液(组分B)或生长培养基中。注意:TF2-VAD-FMK是荧光的;因此,重要的是洗净任何未结合的试剂以除去背景。

6.可选:可以用DNA染色剂标记细胞(例如碘化丙啶或死细胞的7-AAD)。

7.可选:修复细胞。

8.使用带有530/30 nm滤光片的流式细胞仪(FITC通道)检测荧光强度。

 

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