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。
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荧光团 |
兼容的EFLUXX-ID® 试剂 |
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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 |
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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
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。
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）。
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。
◆产品详情
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应用 |
流式细胞术，荧光显微镜 |
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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。 |
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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 |
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包装 |
100 assays（流式细胞术） |
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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。 |
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储存条件 |
避光。避免反复冻融。 |
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运输条件 |
蓝冰运输 |
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短期储存 |
-20°C |
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长期储存 |
-80°C |
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组分 |
2 瓶，EFLUXX-ID® Gold或EFLUXX-ID® Green检测试剂 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 |
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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。 |
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监管状态 |
RUO – 仅供研究用 |
参考文献
◆产品参考文献
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1. |
Exploiting off-target effects of estrogen deprivation to sensitize estrogen receptor negative breast cancer to immune killing: B. Wolfson, et al.; J. Immunother. Cancer 9, 2258 (2021), 摘要; |
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2. |
MicroRNA-324-5p regulates stemness, pathogenesis and sensitivity to bortezomib in multiple myeloma cells by targeting hedgehog signaling: B. Tang, et al.; Int. J. Cancer 142, 109 (2018), Application(s): Flow Cytometry; multiple myeloma, 摘要; Full Text |
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3. |
Upregulation of FOXM1 in a subset of relapsed myeloma results in poor outcome: C. Gu, et al.; Blood Cancer J. 8, 22 (2018), Application(s): Flow Cytometry; myeloma cells, 摘要; Full Text |
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4. |
Resistin induces multidrug resistance in myeloma by inhibiting cell death and upregulating ABC transporter expression: J. Pang, et al.; Haematologica 102, 1273 (2017), Application(s): Flow Cytometry; myeloma cells, 摘要; Full Text |
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5. |
Hepatocyte SLAMF3 reduced specifically the multidrugs resistance protein MRP-1 and increases HCC cells sensitization to anti-cancer drugs: G. Fouquet, et al.; Oncotarget 7, 32493 (2016), 摘要; Full Text |
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6. |
Intercellular transfer of P-glycoprotein in human blood-brain barrier endothelial cells is increased by histone deacetylase inhibitors: A. Noack, et al.; Sci. Rep. 6, 29253 (2016), Application(s): Flow cytometry of Pgp-EGFP transfer in co-cultured cells, 摘要; Full Text |
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7. |
The influence of a caveolin-1 mutant on the function of P-glycoprotein: C.Y. Lee, et al.; Sci. Rep. 6, 20486 (2016), 摘要; Full Text |
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8. |
RhoGDI2 up-regulates P-glycoprotein expression via Rac1 in gastric cancer cells: Z. Zheng, et al.; Cancer Cell Int. 15, 41 (2015), Application(s): Monitoring of MDR1 functionality by flow cytometry, 摘要; Full Text |
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9. |
Role of NEK2A in human cancer and its therapeutic potentials: J. Xia, et al.; Biomed. Res. Int. 2015, 862461 (2015), 摘要; Full Text |
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Sonic hedgehog-glioma associated oncogene homolog 1 signaling enhances drug resistance in CD44+/Musashi-1+ gastric cancer stem cells: M. Xu, et al.; Cancer Lett. 369, 124 (2015), 摘要; |
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11. |
Drug-induced trafficking of p-glycoprotein in human brain capillary endothelial cells as demonstrated by exposure to mitomycin C: A. Noack, et al.; PLoS One 9, e88154 (2014), Application(s): MDR status by flow cytometry in microvascular endothelial cells, 摘要; 全文 |
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12. |
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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13. |
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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14. |
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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1. |
P-Glycoprotein-mediated resistance to Hsp90-directed therapy is eclipsed by the heat shock response: A.K. McCollum, et al.; Cancer Res. 68, 7419 (2008), 摘要; |
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2. |
Glutathione export during apoptosis requires functional multidrug resistance-associated proteins: C.L. Hammond, et al.; J. Biol. Chem. 282, 14337 (2007), 摘要; |
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3. |
From MDR to MXR: new understanding of multidrug resistance systems, their properties and clinical significance: T. Litman, et al.; Cell Mol. Life Sci. 58, 931 (2001), 摘要; |
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4. |
Increased levels of the multidrug resistance protein in lateral membranes of proliferating hepatocyte-derived cells: H. Roelofsen, et al.; Gastroenterology 112, 511 (1997), 摘要; |
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5. |
How to probe clinical tumour samples for P-glycoprotein and multidrug resistance-associated protein: H.J. Broxterman, et al.; Eur. J. Cancer 32A, 1024 (1996), 摘要; |
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6. |
Methods to detect P-glycoprotein-associated multidrug resistance in patients’ tumors: consensus recommendations: W.T. Beck, et al.; Cancer Res. 56, 3010 (1996), 摘要; |
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7. |
Functional detection of MDR1/P170 and MRP/P190-mediated multidrug resistance in tumour cells by flow cytometry: N. Feller, et al.; Br. J. Cancer 72, 543 (1995), 摘要; |
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8. |
Immunocytochemical observation of multidrug resistance (MDR) p170 glycoprotein expression in human osteosarcoma cells. The clinical significance of MDR protein overexpression: B. Bodey, et al.; Anticancer Res. 15 (6B), 2461 (1995), 摘要; |
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9. |
Fluorescent cellular indicators are extruded by the multidrug resistance protein: L. Homolya, et al.; J. Biol. Chem. 268, 21493 (1993), 摘要; |
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10. |
Polarized efflux of 2’,7’-bis(2-carboxyethyl)-5(6)-carboxyfluorescein from cultured epithelial cell monolayers: G.K. Collington, et al.; Biochem. Pharmacol. 44, 417 (1992), 摘要; |
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11. |
Epithelial secretion of vinblastine by human intestinal adenocarcinoma cell (HCT-8 and T84) layers expressing P-glycoprotein: J. Hunter, et al.; Br. J. Cancer 64, 437 (1991), 摘要; |
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12. |
Intrinsic multidrug resistance phenotype of Chinese hamster (rodent) cells in comparison to human cells: R.S. Gupta; BBRC 153, 598 (1988), 摘要; |