Screen Quest™ Fluorimetric MDR Assay Kit

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.

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.

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

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.