Screen Quest™ No Wash Potassium Channel Assay Kit

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Spectral properties

Correction Factor (260 nm)	1.076
Correction Factor (280 nm)	0.769
Extinction coefficient (cm ^-1 M ^-1)	23430
Excitation (nm)	495
Emission (nm)	516
Quantum yield	0.16¹

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12352200

Overview SDS Protocol

Correction Factor (260 nm)

1.076

Correction Factor (280 nm)

0.769

Extinction coefficient (cm ^-1 M ^-1)

23430

Excitation (nm)

495

Emission (nm)

516

Quantum yield

0.16¹

Potassium (K+) ion channel plays an important role in regulating fundamental biological processes including heart rate, hormone and neurotransmitter secretion, water and electrolyte balance. Potassium ion channel has been considered as drug targets for disease indications including arrhythmia, pain, diabetes, neurological dysfunctions etc. The permeability of Tl+ through K+ channel has been widely used to assay K+ channel. The cells that express K+ channel of interests (e.g. hERG, Kv1.3, Kir2.1, KATP) are pre-loaded with a Tl+ sensitive dye. The dye is non-fluorescent and is permeable to cell membrane. Once inside the cell, the non-fluorescent AM ester dye is cleaved by endogenous esterase into a negatively charges dye that stays inside cells. When a stimulus buffer containing low dose of Tl+ is added to cells, the Tl+ flows across the K+ channel and binds to Tl+ sensitive dye, generating a fluorescent signal. This signal is proportional to the activity of K+ channel. If an antagonist or antagonist is added to the cells, the fluorescent signal decreases or increases respectively, to reflect the inhibited or stimulated activity of K+ channel.

Platform

Fluorescence microplate reader

Excitation	490 nm
Emission	525 nm
Cutoff	515 nm
Recommended plate	Black wall/clear bottom
Instrument specification(s)	Bottom read mode/Programmable liquid handling

Other instruments

ArrayScan, FDSS, FLIPR, FlexStation, IN Cell Analyzer, NOVOStar, ViewLux

Components

Example protocol

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/K₂SO₄Stimulus 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

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.

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: Tl₂SO₄is 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-KCNH₂cells), 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.

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Correction Factor (260 nm)	1.076
Correction Factor (280 nm)	0.769
Extinction coefficient (cm ^-1 M ^-1)	23430
Excitation (nm)	495
Emission (nm)	516
Quantum yield	0.16¹

Images

Figure 1. 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.

Figure 2. K₂SO₄dose dependent hERG channel activity was measured in HEK293-KCNH₂ cells with Screen Quest^TMPotassium 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 Tl₂SO₄and different concentration of K₂SO₄containing 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.