多药耐药性检测试剂盒——监测三种ABC转运蛋白 EFLUXX-ID® multidrug resistance assay kit

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EFLUXX-ID® multidrug resistance assay kit多药耐药性检测试剂盒——监测三种ABC转运蛋白                              EFLUXX-ID® multidrug resistance assay kit

多药耐药性检测试剂盒——监测三种ABC转运蛋白

简单免洗检测,可同时监测3种耐药性相关的ABC转运蛋白

耐药性是一种因ATP结合盒式(ATP Binding Cassette,ABC)膜转运蛋白家族上调介导所产生的现象。

ABC转运蛋白的过度表达了加速有毒物质从细胞中的去除,例如化疗剂从肿瘤细胞中排出,或抗生素从耐药菌株中排出。

Enzo的EFLUXX-ID® 多药耐药性检测试剂盒可实现同时对三种临床相关的 ABC 转运蛋白的功能检测:包括MDR1(p-糖蛋白)、MRP1/2 和 BCRP。

 

◆特点

● 简单免洗的操作步骤,结果可在1小时内获得

● 可检测与3种ABC转运蛋白(MDR1(p-糖蛋白)、MRP1/2和BCRP)活性相关的耐药性

● 通过单一专用染料可定量检测活细胞中的多药耐药性(MDR)活性,并以MDR 活性因子(MAF)表征

● 试剂盒内含有三种已知的MDR1(p-糖蛋白)、MRP1/2和BCRP蛋白特异性抑制剂

● 可检测Calcein AM染料无法检测的BCRP蛋白活性

● 有绿色、金色两种荧光染料可供选择

● 两种染料均可与表达GFP的细胞系或其他CELLESTIAL® 染料同时使用


作用机制


 EFLUXX-ID®使用了一种疏水性非荧光化合物,这种化合物容易穿透细胞膜,经细胞内酯酶水解成亲水性荧光染料。除非EFLUXX-ID® 染料被泵出至细胞外,否则酯酶裂解的染料会一直存在于细胞内。因此,表现出耐药性的细胞将染料主动运输泵出胞外,会出现荧光减弱的现象。EFLUXX-ID® 检测是目前少有可同时监测三种主要的ABC转运蛋白并能够分析单泵活性的试剂盒。

  荧光团

  兼容的EFLUXX-ID® 试剂

  Cy 3 & Cy 5的偶联物、Pl、7-AAD、APC、Texas Red、

  RFP、YFP、TAMRA、UV和Violet 染料

  EFLUXX-ID® 绿色荧光染料

  GFP/eGFP、RFP、PI、7-AAD、APC、Cy 5的偶联物、

  Rhodamine   123、Texas Red、UV和Violet   染料

  EFLUXX-ID® 金色荧光染料

表1:EFLUXX-ID® 多药耐药性染料与其他荧光染料的兼容性

多药耐药性检测试剂盒——监测三种ABC转运蛋白                              EFLUXX-ID® multidrug resistance assay kit

图1:对三种主要ABC转运蛋白的活性进行检测。使用EFLUXX -ID Green(上)、Gold(中)或Calcein AM(下)染料,通过流式细胞术评估CHO K1细胞中ABC转运蛋白的活性。与未处理的细胞相比,用ABC转运蛋白特异性抑制剂(图中阴影部分)处理可诱导染料保留在细胞内(图中实线部分)。平均荧光强度(MFI)的差异表明了对应蛋白的活性,在图中通过多药耐药性活性因子值(MAF)进行表征。更高的MAF值表明EFLUXX-ID染料对抑制剂有着良好的特异性。而Calcein AM染料的低MAF值表明其(常用的MDR检测探针)无法检测BCRP活性。

多药耐药性检测试剂盒——监测三种ABC转运蛋白                              EFLUXX-ID® multidrug resistance assay kit

图2:使用EFLUXX-ID® Green和EFLUXX-ID® Gold染料在CHO K1细胞中评估已知抑制剂对ABC转运蛋白活性的结果分析。将细胞与试剂盒中含有的MDR普通抑制剂(左一)、转运蛋白特异性抑制剂在37°C下孵育5 min。随后在37°C下用指定的染料染色细胞30 min,并立即利用流式细胞术进行分析。所用抑制剂:5 µM环胞霉素 A(MDR普通抑制剂)、20 µM维拉帕米(P-gp特异性抑制剂)、0.05 mM MK-571(MRP特异性抑制剂)、0.05 mM新生霉素(BCRP特异性抑制剂)。

多药耐药性检测试剂盒——监测三种ABC转运蛋白                              EFLUXX-ID® multidrug resistance assay kit

图3:EFLUXX-ID® Green 490/514 nm ex/em和Gold 530/570 nm ex/em 试剂的光谱特性可与其他常见的荧光染料进行多重检测。

多药耐药性检测试剂盒——监测三种ABC转运蛋白                              EFLUXX-ID® multidrug resistance assay kit

◆产品详情


  应用

  流式细胞术,荧光显微镜

  应用说明

  EFLUXX-ID® 多药耐药性检测试剂盒可对活细胞(悬浮细胞和贴壁细胞)中的耐药表型进行检测和分析。

  品质保证

  每批次EFLUXX-ID® Gold耐药检测试剂盒的样品试剂盒均已按照操作手册中的步骤,对第5代和第20代间的

  CHO K1细胞系进行染色。获得的结果如下

  1.   维拉帕米介导的抑制后MAF>80

  2. MK-571介导的抑制后MAF>60

  3. 新生霉素介导的抑制后MAF>40

  包装

  100 assays(流式细胞术)

  使用/稳定性

  自交货后,试剂盒组分在适当的储存条件下可稳定保存一年。

  储存条件

  避光。避免反复冻融。

  运输条件

  蓝冰运输

  短期储存

  -20°C

  长期储存

  -80°C

  组分

  2 瓶,EFLUXX-ID® Gold或EFLUXX-ID® Green检测试

  300 nmoles,MDR1 抑制剂(维拉帕米)

  750 nmoles,MRP   抑制剂(MK-571)

  1.5 µmoles,BCRP 抑制剂(新生霉素)

  500 µL,碘化丙啶

  科学背景

  耐药性是一种由跨膜ATP结合盒(ATP   Binding Cassette   ,简称ABC)转运蛋白家族上调介导的现象。

  ABC转运蛋白的过表达加速了毒性剂从细胞中去除,例如化疗剂从肿瘤细胞中流出,或抗生素从耐药菌株中流出。

  监管状态

  RUO – 仅供研究用

参考文献



◆产品参考文献


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NEK2 induces drug resistance mainly through activation of efflux drug pumps and is associated with poor prognosis in myeloma and other cancers: W. Zhou, et al.; Cancer Cell 23, 48 (2013), Application(s): Dye Efflux Assay for Multidrug Resistance was performed with ARP1 cancer cell lines and MCF7 cells using Gold detection reagent on flow cytometry, 摘要;


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Localization microscopy (SPDM) reveals clustered formations of P-glycoprotein in a human blood-brain barrier model: O. Huber, et al.; PLoS One 7, e44776 (2012), Application(s): MDR status by flow cytometry in immortalized human brain endothelial cells, 摘要全文


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Sensitive and specific fluorescent probes for functional analysis of the three major types of mammalian ABC transporters.: I. Lebedeva, et al.; PLoS One 6, e22429 (2011), 摘要全文

◆其他文献


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