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EFLUXX-ID® Gold multidrug resistance assay kit

Simple no-wash assays for simultaneous monitoring of all 3 major  ABC transporter proteins – MDR, BCRP & MRP
 
ENZ-51030-K100 1 Kit 538.00 USD
Do you need bulk/larger quantities?
 
  • Measures drug resistance related to activity of all three major ABC transporter proteins.
  • Single proprietary dye provides quantitative measurements of MDR activity in live cells expressed as MDR Activity Factor (MAF).
  • Kit includes three known inhibitors specific for MDR1 (p-glycoprotein), MRP1/2, and BCRP.
  • Simple, no-wash protocol delivers results in 1 hour.
  • Available in green and gold fluorophores.
  • Comprehensive efflux detection assay for three major types of ABC transporters.
  • Detects BCRP activity not detected with Calcein AM.
  • Inhibitors included for profiling of specific pumps involved in drug resistance.
  • Two dye formats allows multiplexing with GFP-expressing cell lines or other CELLESTIAL® dyes.
The EFLUXX-ID® Multidrug Resistance Assay Kit allows for functional detection of all three clinically relevant ABC transporter proteins: MDR1 (p-glycoprotein), MRP1/2, and BCRP. The assay uses a hydrophobic, non-fluorescent compound that readily penetrates the cell membrane, where it is hydrolyzed to a hydrophilic fluorescent dye by intracellular esterases. Unless the EFLUXX-ID® dye is pumped out of the cell, the esterase cleaved dye is trapped inside the cell. Thus, cells exhibiting drug resistance will have diminished fluorescence. EFLUXX-ID® assay is the only available kit for the simultaneous monitoring of all three major ABC reporter proteins with the ability to profile individual pump activity.

Mechanism of Action
The proprietary AM-ester form of the EFLUXX-ID® dye is a hydrophobic non-fluorescent compound that readily penetrates the cell membrane and is subsequently hydrolyzed inside of the cells by intracellular esterases. Unless the EFLUXX-ID® dye is pumped out of the cell, the esterase cleaved dye is trapped inside the cell. The fluorescence signal of the dye generated within the cells thus depends upon the activity of the ABC transporters. The cells with highly active transporters will demonstrate lower fluorescence because of the active efflux of the probe from the cell. Application of specific inhibitors of the various ABC transporter proteins allows differentiation between the three common types of pumps.
MOA ENZ-51029-30 webimage 01
ENZ-51029-30 Data-1 webimage 01
Figure 1: Detect Activity of all three major ABC transporter proteins. ABC transporter protein activity was evaluated in CHO K1 cells by flow cytometry using eFluxx-ID Green (top), Gold (middle), or Calcein AM (bottom) dyes. Treatment with specific inhibitors of ABC Transporter proteins (shaded histograms) induces retention of dye within cells relative to untreated cells (lined histograms). The difference in mean fluorescence intensity (MFI) is an indication of the corresponding protein activity, as shown by MAF scores [multidrug resistance activity factors], a quantitative measurement of multidrug resistance. Higher MAF scores are a result of superior specificity of eFluxx-ID dyes to specific inhibitors. Calcein AM (a common probe for MDR assays), is unable to detect BCRP activity.
ENZ-51029-30 Data-2 webimage 01
Figure 2: Profiling of ABC transporter activity by known inhibitors was assessed in CHO K1 cells using eFluxx-ID Green and eFluxx-ID Gold dyes. Cells were incubated for 5 min at 37°C with general MDR Inhibitor (far left column) or transporter-specific inhibitors included in the kit. Cells were then loaded with the indicated dye for 30 min at 37°C and immediately analyzed by flow cytometry. Inhibitors used: 5 µM Cyclosporin A (general MDR inhibitor), 20 µM Verapamil (specific P-gp inhibitor), 0.05 mM MK-571 (specific MRP inhibitor), 0.05 mM Novobiocin (specific BCRP inhibitor).
Green-Gold Spectra webimage 01
Figure 3: Spectral characteristics of the eFluxx-ID Green 490/514 nm ex/em and Gold 530/570 nm ex/em reagents allows for multiplexing with other common fluorescent dyes.
ENZ-51029-30 Table 1 webimage 01
Table 1: Compatibility of eFluxx-ID MDR dyes with other fluorescent dyes.
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MOA ENZ-51029-30 webimage 01 ENZ-51029-30 Data-1 webimage 01 ENZ-51029-30 Data-2 webimage 01 Green-Gold Spectra webimage 01 ENZ-51029-30 Table 1 webimage 01

Product Specification

Applications:Flow Cytometry, Fluorescence microscopy
 
Application Notes:The EFLUXX-ID® Gold multidrug resistance assay kit is designed for functional detection and profiling of multidrug resistant phenotypes in live cells (both suspension and adherent).
 
Quality Control:A sample kit from each lot of EFLUXX-ID® Gold multidrug resistance assay kit is used to stain CHO K1 cell line which is used between passages 5 and 20, using the procedures described in the user manual. The following results were obtained:
1. MAF >80 after verapamil-mediated inhibition
2. MAF >60 after MK-571-mediated inhibition
3. MAF >40 after novobiocin-mediated inhibition
 
Quantity:100 assays. For flow cytometry.
 
Use/Stability:With proper storage, the kit components are stable for one year upon receipt.
 
Handling:Protect from light. Avoid freeze/thaw cycles.
 
Shipping:Shipped on Dry Ice
 
Short Term Storage:-20°C
 
Long Term Storage:-80°C
 
Kit/Set Contains:2 vials, EFLUXX-ID® Gold Detection Reagent
300 nmoles, MDR1 Inhibitor (Verapamil)
750 nmoles, MRP Inhibitor (MK-571)
1.5 µmoles, BCRP Inhibitor (Novobiocin)
500 µl, Propidium Iodide
 
Scientific Background:Drug resistance is a phenomenon mediated by up-regulation of a family of transmembrane ATP Binding Cassette (ABC) transporter proteins. Overexpression of ABC transporter proteins accelerates removal of toxic agents from the cell, for example the efflux of chemotherapeutic agents from tumor cells, or of antibiotics from resistant strains of bacteria.
 

Product Literature References

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, Abstract; Full Text
The influence of a caveolin-1 mutant on the function of P-glycoprotein: C.Y. Lee, et al.; Sci. Rep. 6, 20486 (2016), Abstract; Full Text
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, Abstract; Full Text
Role of NEK2A in human cancer and its therapeutic potentials: J. Xia, et al.; Biomed. Res. Int. 2015, 862461 (2015), Abstract; Full Text
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), Abstract;
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, Abstract; Full Text
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, Abstract;
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, Abstract; Full Text
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), Abstract; Full Text

General Literature References

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), Abstract;
Glutathione export during apoptosis requires functional multidrug resistance-associated proteins: C.L. Hammond, et al.; J. Biol. Chem. 282, 14337 (2007), Abstract;
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), Abstract;
Increased levels of the multidrug resistance protein in lateral membranes of proliferating hepatocyte-derived cells: H. Roelofsen, et al.; Gastroenterology 112, 511 (1997), Abstract;
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), Abstract;
Methods to detect P-glycoprotein-associated multidrug resistance in patients’ tumors: consensus recommendations: W.T. Beck, et al.; Cancer Res. 56, 3010 (1996), Abstract;
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), Abstract;
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), Abstract;
Fluorescent cellular indicators are extruded by the multidrug resistance protein: L. Homolya, et al.; J. Biol. Chem. 268, 21493 (1993), Abstract;
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), Abstract;
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), Abstract;
Intrinsic multidrug resistance phenotype of Chinese hamster (rodent) cells in comparison to human cells: R.S. Gupta; BBRC 153, 598 (1988), Abstract;

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EFLUXX-ID® Green multidrug resistance assay kit 

Simple No-wash Assay for  Simultaneous Monitoring of All 3 Major ABC Transporter Proteins – MDR, BCRP & MRP
Flow Cytometry, Fluorescence microscopy | Print as PDF
 
ENZ-51029-K100 1 Kit 538.00 USD
Do you need bulk/larger quantities?
 

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