Screen Quest™ Fluorimetric MDR Assay Kit

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3e+42e+42e+48.0e+310001001010.11e-2- Dose-responseData legend Generated with Quest Graph™ Cyclosporin (uM) RFU Hover mouse to interact
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Unit Size: Cat No: Price (USD): Qty:
36341 $950


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Additional Ordering Information
Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
International: See distributors





Overview

PlatformsFluorescence microplate reader
Storage Freeze (<-15 °C)
Minimize light exposure
Category ADME and Tox
MDR Research
Related Cell Metabolism
Cell Functional Analysis
Tumor cell resistance to cytotoxic drugs is considered one of the major obstacles to successful chemotherapy. Some tumors are initially resistant and never respond to cytostatic drug treatment; others initially respond well but eventually regrow and become resistant. This phenomenon may result from genetic mutations induced by the administered antitumor agent, or may represent the selection of preexisting resistant cell populations in the malignant tumor. Multi-drug resistance (MDR) is a major factor in the failure of many forms of chemotherapy. In the past few years it has become widely accepted that the resistance to chemotherapy correlates with the overexpression of at least two ATP-dependent drug-efflux pumps. These cell membrane proteins, called P-glycoprotein (Pgp, MDR1), and multidrug-resistance-associated protein (MRP1) are members of the ABC transporter family. Our assay kit uses a fluorescent MDR indicator for assaying these two MDR pump activities. This hydrophobic fluorescent dye molecule rapidly penetrates cell membranes and becomes trapped in cells. Following a short incubation, the intracellular free dye concentration can increase significantly. In the MDR1 and/or MRP1-expressing cells this dye is extruded by the MDR transporter, thus decreasing the cellular fluorescence intensity. However, when its extrusion is blocked by an agent that interferes with the MDR1 and/or MRP1 pump-activity, its cellular fluorescence intensity increases significantly. Our MDR assay kit provides all the essential components with an optimized assay method. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation. This assay kit is ideal for high throughput screening of MDR pump inhibitors or identifying the cells that have high level of MDR pump activities.




Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

Protocol summary

  1. Prepare cells
  2. Add MDR inhibitors or compounds
  3. Add MDR dye-loading solution (100 µL/well for 96-well plate or 25 µL/well for 384-well plate)
  4. Incubate at room temperature for 1 hour
  5. Monitor fluorescence intensity at Ex/Em = 490/525 nm with bottom read mode

Important notes
Thaw all the kit components at room temperature before use.

Key parameters
Instrument:Fluorescence microplate reader
Excitation:490 nm
Emission:525 nm
Cutoff:515 nm
Recommended plate:Solid black
Preparation of stock solution
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

MDR sensor stock solution:
Add 20 µL (Cat. # 36340-1 plate) or 200 µL (Cat. # 36341-10 plates) of DMSO (Component B) into MDR sensor (Component A), and mix them well. Note: 20 µL of MDR sensor stock solution is enough for one plate. Un-used MDR sensor stock solution can be aliquoted and stored at < -20 oC for one month if the tubes are sealed tightly. Protect from light and avoid repeated freeze-thaw cycles and moisture.

Preparation of working solution

MDR dye-loading solution:
Add 20 µL of MDR sensor stock solution into 10 mL of Assay Buffer (Component C), and mix them well. Note: The MDR dye-loading solution is enough for one plate and stable for at least 2 hours at room temperature.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

Sample experimental protocol
  1. Treat cells with test compounds by adding 10 µL of 10X (96-well plate) or 5 µL of 5X (384-well plate) compounds into compound buffer (such as PBS or HHBS). For blank wells (medium without the cells), add the corresponding amount of compound buffer. Note: It is not necessary to wash cells before adding compound. However, if tested compounds are serum sensitive, growth medium and serum factors can be aspirated away before adding compounds. Add the same volume of HHBS into the wells (such as 90 µL for a 96-well plate or 20 µL for a 384-well plate) after aspiration. Alternatively, cells can be grown in serum-free media.

  2. Incubate the cell plate at room temperature or in a 37 oC, 5% CO2 incubator for at least 15 minutes or a desired period of time.

  3. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of MDR dye-loading solution.

  4. Incubate the dye-loading plate at room temperature for 1 hour, protected from light. Note: The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment. (We got the optimal results with the incubation time less than 4 hours.) Note: DO NOT wash the cells after loading. Note: For non-adherent cells, it is recommended to centrifuge the cell plate at 800 rpm for 2 minutes with brake off after incubation.

  5. Monitor the fluorescence intensity at Ex/Em = 490/525 nm with bottom read mode.
Example data analysis and figures

The reading (RFU) obtained from the blank standard well is used as a negative control. Subtract this value from the other standards' readings to obtain the base-line corrected values. Then, plot the standards' readings to obtain a standard curve and equation. This equation can be used to calculate Cyclosporin samples. We recommend using the Online Four Parameter Logistics Calculator which can be found at:

https://www.aatbio.com/tools/four-parameter-logistic-4pl-curve-regression-online-calculator

Figure 1. Effect of Cyclosporin A on the inhibition of P-gp pump in MCF-7/ADR cells. The increased concentration of Cyclosporin A resulted in an increase in fluorescence signal caused by the inhibition of P-gp pump which enhanced the intracellular accumulation of MDR indicator dye. The EC50 = 2.4 μM (measured with the kit) is similar to the value reported in the literature.

Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





References

A phase I clinical and pharmacokinetic study of the multi-drug resistance protein-1 (MRP-1) inhibitor sulindac, in combination with epirubicin in patients with advanced cancer
Authors: O'Connor R, O'Leary M, Ballot J, Collins CD, Kinsella P, Mager DE, Arnold RD, O'Driscoll L, Larkin A, Kennedy S, Fennelly D, Clynes M, Crown J.
Journal: Cancer Chemother Pharmacol (2007): 79

Mutational Patterns Associated with the 69 Insertion Complex in Multi-drug-resistant HIV-1 Reverse Transcriptase that Confer Increased Excision Activity and High-level Resistance to Zidovudine
Authors: Cases-Gonzalez CE, Franco S, Martinez MA, Menendez-Arias L.
Journal: J Mol Biol (2007): 298

Activity-guided isolation of scopoletin and isoscopoletin, the inhibitory active principles towards CCRF-CEM leukaemia cells and multi-drug resistant CEM/ADR5000 cells, from Artemisia argyi
Authors: Adams M, Efferth T, Bauer R.
Journal: Planta Med (2006): 862

Assessment of resistance in multi drug resistant tuberculosis patients
Authors: Irfan S, Hassan Q, Hasan R.
Journal: J Pak Med Assoc (2006): 397

Clinical Prediction Tool to Identify Patients with Pseudomonas aeruginosa Respiratory Tract Infections at Greatest Risk for Multi-Drug Resistance
Authors: Lodise TP, Miller CD, Graves J, Furuno JP, McGregor JC, Lomaestro B, Graffunder E, McNutt LA.
Journal: Antimicrob Agents Chemother. (2006)

Colistin, meropenem and rifampin in a combination therapy for multi-drug-resistant Acinetobacter baumannii multifocal infection
Authors: Biancofiore G, Tascini C, Bisa M, Gemignani G, Bindi ML, Leonildi A, Giannotti G, Menichetti F.
Journal: Minerva Anestesiol. (2006)

Computational simulations of HIV-1 proteases-multi-drug resistance due to nonactive site mutation L90M
Authors: Ode H, Neya S, Hata M, Sugiura W, Hoshino T.
Journal: J Am Chem Soc (2006): 7887

Detection of Multi-Drug Resistance in Mycobacterium tuberculosis
Authors: Sekiguchi JI, Miyoshi-Akiyama T, Augustynowicz-Kopec E, Zwolska Z, Kirikae F, Toyota E, Kobayashi I, Morita K, Kudo K, Kato S, Kuratsuji T, Mori T, Kirikae T.
Journal: J Clin Microbiol. (2006)

Durable efficacy of tipranavir-ritonavir in combination with an optimised background regimen of antiretroviral drugs for treatment-experienced HIV-1-infected patients at 48 weeks in the Randomized Evaluation of Strategic Intervention in multi-drug reSistant patients with Tipranavir (RESIST) studies: an analysis of combined data from two randomised open-label trials
Authors: Hicks CB, Cahn P, Cooper DA, Walmsley SL, Katlama C, Clotet B, Lazzarin A, Johnson MA, Neubacher D, Mayers D, Valdez H.
Journal: Lancet (2006): 466

Editorial: the treatment of multi-drug resistant tuberculosis--a return to the pre-antibiotic era
Authors: Olle-Goig JE.
Journal: Trop Med Int Health (2006): 1625


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