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	478.00 USD

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. The resulting fluorescent dye is trapped inside the cell unless it is actively pumped out by ABC transporter protein activity.

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.

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).

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.

Table 1: Compatibility of eFluxx-ID MDR dyes with other fluorescent dyes.

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Product Specification

Quantity:	100 assays. For flow cytometry.

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 of uptake 2. MAF >60 after MK-571-mediated inhibition of uptake 3. MAF >40 after novobiocin-mediated inhibition of uptake

Kit/Set Contains:	1 vial, 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

Application:	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).

Short Term Storage:	-20°C

Long Term Storage:	-80°C

Use/Stability:	With proper storage, the kit components are stable for one year upon receipt.

Handling:	Avoid freeze/thaw cycles. Protect from light.

Background / Technical Information:	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

Sensitive and specific fluorescent probes for functional analysis of the three major types of mammalian ABC transporters.: I. Lebedeva, et al.; PLoS One 6 (7), (2011), Abstract;

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;