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Screen Quest™ Fluorimetric MDR Assay Kit

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.
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.
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.
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H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
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OverviewpdfSDSpdfProtocol


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.

Platform


Fluorescence microplate reader

Excitation490 nm
Emission525 nm
Cutoff515 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode

Components


Example protocol


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.

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.

Images


References


View all 77 references: Citation Explorer
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
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
Identification of multi-drug resistant Pseudomonas aeruginosa clinical isolates that are highly disruptive to the intestinal epithelial barrier
Authors: Zaborina O, Kohler JE, Wang Y, Bethel C, Shevchenko O, Wu L, Turner JR, Alverdy JC.
Journal: Ann Clin Microbiol Antimicrob (2006): 14
Therapeutic significance of ectophosphatase inhibitors in reversal of multi-drug resistance
Authors: Kannan S., undefined
Journal: Med Hypotheses (2006): 1041
Newer diagnostics for tuberculosis and multi-drug resistant tuberculosis
Authors: Palomino JC., undefined
Journal: Curr Opin Pulm Med (2006): 172
Tipranavir: a protease inhibitor for multi-drug resistant HIV-1
Authors: Best B, Haubrich R.
Journal: Expert Opin Investig Drugs (2006): 59
Long-term persistence of multi-drug-resistant Salmonella enterica serovar Newport in two dairy herds
Authors: Cobbold RN, Rice DH, Davis MA, Besser TE, Hancock DD.
Journal: J Am Vet Med Assoc (2006): 585
Bacteriologic profile of bacteremia due to multi-drug resistant bacteria at Charles-Nicolle Hospital of Tunis
Authors: Saidani M, Boutiba I, Ghozzi R, Kammoun A, Ben Redjeb S.
Journal: Med Mal Infect (2006): 163
Reversal of multi-drug resistance in ovarian cancer cell by RNA interference
Authors: Lou JY, Peng ZL, Zheng Y, Wang H, He B, Wang HJ.
Journal: Zhonghua Fu Chan Ke Za Zhi (2006): 413
Regulation of MDK expression in human cancer cells modulates sensitivities to various anticancer drugs: MDK overexpression confers to a multi-drug resistance
Authors: Kang HC, Kim IJ, Park HW, Jang SG, Ahn SA, Yoon SN, Chang HJ, Yoo BC, Park JG.
Journal: Cancer Lett. (2006)