Screen Quest™ No Wash Potassium Channel Assay Kit
![Astemizole dose dependent inhibition of hERG channel was measured in HEK293-KCNH2 cells with Screen Quest™ Potassium Ion Channel Kit. The cells were seeded overnight at 20,000 cells/100 µL/well in a Costar black wall/clear bottom 96-well poly-D-lysine plate. The cells were incubated with 100 µL of dye-loading solution for 1 hour at 37°C. 10 µL of Astemizole was added to the cells and incubated for 30 minutes at 37°C. 50 µL of 0.5 mM Tl2SO4 and 2.5 mM K2SO4 containing stimulus solution was injected in each well by FlexStation and read every sec for 3 minutes at excitation/emission=490/525nm. IC50 = 0.46 µM.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fscreen-quest-no-wash-potassium-channel-assay-kit%2Fgraph-for-screen-quest-no-wash-potassium-channel-assay-kit_Apz1o.webp&w=640&q=75)
![Astemizole dose dependent inhibition of hERG channel was measured in HEK293-KCNH2 cells with Screen Quest™ Potassium Ion Channel Kit. The cells were seeded overnight at 20,000 cells/100 µL/well in a Costar black wall/clear bottom 96-well poly-D-lysine plate. The cells were incubated with 100 µL of dye-loading solution for 1 hour at 37°C. 10 µL of Astemizole was added to the cells and incubated for 30 minutes at 37°C. 50 µL of 0.5 mM Tl2SO4 and 2.5 mM K2SO4 containing stimulus solution was injected in each well by FlexStation and read every sec for 3 minutes at excitation/emission=490/525nm. IC50 = 0.46 µM.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fscreen-quest-no-wash-potassium-channel-assay-kit%2Fgraph-for-screen-quest-no-wash-potassium-channel-assay-kit_Apz1o.webp&w=640&q=75)
![Astemizole dose dependent inhibition of hERG channel was measured in HEK293-KCNH2 cells with Screen Quest™ Potassium Ion Channel Kit. The cells were seeded overnight at 20,000 cells/100 µL/well in a Costar black wall/clear bottom 96-well poly-D-lysine plate. The cells were incubated with 100 µL of dye-loading solution for 1 hour at 37°C. 10 µL of Astemizole was added to the cells and incubated for 30 minutes at 37°C. 50 µL of 0.5 mM Tl2SO4 and 2.5 mM K2SO4 containing stimulus solution was injected in each well by FlexStation and read every sec for 3 minutes at excitation/emission=490/525nm. IC50 = 0.46 µM.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fscreen-quest-no-wash-potassium-channel-assay-kit%2Fgraph-for-screen-quest-no-wash-potassium-channel-assay-kit_Apz1o.webp&w=128&q=25)
![K<sub>2</sub>SO<sub>4 </sub>dose dependent hERG <sup> </sup>channel activity was measured in HEK293-KCNH<sub>2</sub> cells with Screen Quest<sup>TM </sup>Potassium Ion Channel Kit. The cells were seeded overnight at 20,000 cells/100 µL/well in a Costar black wall/clear bottom 96-well poly-D-lysine plate. The cells were incubated with 100 µL of dye-loading solution for 1 hour at 37°C. Final concentration of 1 mM , 0.5 mM, 0.25 mM or 0 mM Tl<sub>2</sub>SO<sub>4 </sub>and different concentration of K<sub>2</sub>SO<sub>4 </sub>containing stimulus solution was injected in each well by FlexStation and the plate was read every 1-2 sec for 3 minutes at excitation/emission=490/525nm.](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fproducts%2Ffigures-and-data%2Fscreen-quest-no-wash-potassium-channel-assay-kit%2Ffigure-for-screen-quest-no-wash-potassium-channel-assay-kit_PeLKy.jpg&w=128&q=25)
AT A GLANCE
Protocol summary
- Prepare cells in growth medium
- Add Tl-520 AM dye-loading solution (100 µL/well for 96-well plate or 25 µL/well for 384-well plate)
- Incubate at 37°C for 1 hour
- Add agonist/antagonist and incubate at 37 °C for 30 minutes
- Add Tl2SO4/K2SO4 Stimulus solution (50 µL/well for 96-well plate or 12.5 µL/well for 384-well plate)
- Monitor fluorescence intensity at Ex/Em = 490/525 nm
Important notes
Do not add additional probenecid. Thaw all the kit components at room temperature before use.
PREPARATION OF STOCK SOLUTION
1. TI-520 AM stock solution:
Add 20 µL (Cat. # 36550) or 200 µL of (Cat. # 36551 and # 36552) of DMSO into the vial of TI-520 AM (Component A), and mix them well. Note: The unused TI-520 AM stock solution can be aliquoted and stored at < -20 oC. Protect from light and avoid repeated freeze-thaw cycles.
2. Assay Buffer (1X):
Add 1 mL of 10X Pluronic® F127 Plus (Component B) into 9 mL of HHBS buffer (Component C) and mix well. Note: 10 mL of 1X assay buffer is enough for one plate. Aliquot and store un-used 10X Pluronic® F127 Plus (Component B) at < -20 oC. Protect from light and avoid repeated freeze-thaw cycles.
3. Chloride Free Buffer (1X):
Add 2 mL of Chloride Free Buffer (5X) (Component D) in 8 mL of ddH2O, and mix them well.
4. Tl2SO4 solution (Not provided, Sigma Cat# 208191):
Dissolve Tl2SO4 in ultrapure H2O to final concentration of 80 mM. Note: Tl2SO4 is toxic. Take necessary precautions to prevent inhalation and skin contact.
PREPARATION OF WORKING SOLUTION
1. TI-520 AM dye-loading solution:
Add 20 µL of TI-520 AM stock solution (500X) into 10 mL of 1X Assay Buffer and mix well.
2. Stimulus Solution(5X):
Dilute ligands (for non-voltage gated potassium channels) or K2SO4 (for voltage gated potassium channels) with Tl2SO4 in 1X Chloride-free buffer.
Table 1. Reference (for voltage gated hERG channel in HEK293-KCNH2 cells), concentration of Tl2SO4 and K2SO4 (for voltage gated potassium channels) or ligands (for non-voltage gated potassium channels) used for the assay should be optimized for each target channel and cell type.
Components |
Volume |
5X Conc |
Final Conc |
Chloride-free Buffer(5X) |
2 mL |
|
|
K2SO4 (250 mM) |
1 mL |
25 mM |
5 mM |
TI2SO4 (80mM) |
0.5 mL |
4 mM |
0.8 mM |
ddH2O |
5.5 mL |
|
|
Total: |
10 mL |
|
|
For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html
SAMPLE EXPERIMENTAL PROTOCOL
- Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of TI-520 AM dye-loading solution into the cell plate.
- Incubate the dye-loading plate in a cell incubator for 1 hour, and then incubate the plate at room temperature for another 15 to 30 minutes. Note: If the assay requires 37°C, perform the experiment immediately without further room temperature incubation.
- Prepare the K+ channel antagonists or agonists with HHBS.
- Incubate the plate with antagonists (for inhibitory study) in the cell incubator for 30 minutes.
- Add 50 µL/well (for a 96-well plate) or 12.5 µL/well (for a 384-well plate) of 5X stimulus buffer with FLIPR, FDSS or Flexstation. Run the experiment with a filter set of Ex/Em = 490/525 nm. Read the plate every 1–2 seconds for 3 minutes.