Confirmatory screen for compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1
Assay Implementation: Meng Wu Ph.D., Amy Scott M.S., Shunyou Long M.S., Haibo Yu Ph.D., Beiyan Zou Ph.D., Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D. ..more
BioActive Compounds: 927
Data Source: Johns Hopkins Ion Channel Center (JHICC_Kir2.1_Confirm_1)
BioAssay Type: Other, Duplicate, Single Concentration Activity Observed.
Source (MLPCN Center Name): Johns Hopkins Ion Channel Center (JHICC)
Center Affiliation: Johns Hopkins University, School of Medicine
Screening Center PI: Min Li, Ph.D.
Assay Provider: Elena Makhina Ph.D., University of Pittsburgh
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 DA026212-01
Grant Proposal PI: Elena Makhina Ph.D., University of Pittsburgh
Assay Implementation: Meng Wu Ph.D., Amy Scott M.S., Shunyou Long M.S., Haibo Yu Ph.D., Beiyan Zou Ph.D., Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D.
Name: Confirmatory screen for compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1
See the related essay (PubChem AID: 1672).
The purpose of this assay is to validate compounds identified in the primary screen assay (PubChem AID 1672). It employs the same experimental conditions as presented in the primary screen assay. Compounds were tested in duplicates and their effects were evaluated by the calculated FluxOR fluorescence ratio percentage, normalized with negative controls without compounds. If the compound causes 3SD (of negative controls) or more activity decrease (of negative controls) in both duplicates, and is active in the primary screen, the compound is considered to be active that inhibits/blocks inward rectifying potassium ion channel Kir2.1 is considered to be active.
Protocol for the Kir2.1 project:
1. Cell culture: Cells are routinely cultured in DMEM/F12 medium, supplemented with 10% Fetal Bovine Serum (FBS), 50 IU/ml penicillin, 50ug/ml streptomycin, and 500ug/ml G418.
2. Cell plating: Add 50 ul/well of 300,000 cells/ml re-suspended in DMEM/F12 medium with 10% FBS.
3. Incubate overnight at 37C and 5% CO2.
4. Remove medium and add 25 ul/well of 1x FluxOR solution to cells.
5. Incubate 90 minutes, at room temperature (RT), in the dark.
6. Prepare 7.5X compound plates and control plates on the Cybi-Well system: test compounds are prepared using assay buffer; controls are assay buffer (IC0), and IC100 of Chlorpromaizne (all with DMSO concentrations matched to that of test compounds) .
7. Remove FluxOR dye solution and add 20 ul/well of assay buffer to cells.
8. Add 4 ul of 7.5x compound stock into the cell plates via the Cybi-Well system.
9. Incubate all cell plates for 20 minutes at RT in the dark.
10. Prepare 5x stimulus buffer containing 25 mM K2SO4 and 7 mM Tl2SO4.
11. Load cell plates to Hamamatsu FDSS 6000 kinetic imaging plate reader.
12. Measure fluorescence for 10 seconds at 1Hz to establish baseline.
13. Add 6 ul/well of stimulus buffer onto cells and continue measuring fluorescence for 110 seconds.
14. Calculate ratio readout as F(max-min)/F0.
15. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z' factors.
16. Calculate the percentages of tested compounds with the following formula:
Percentage (%) = (Ratio(cpd)- Ratio(Blank))/(Ratio(NC) - Ratio(Blank))*100
Percentage (%): Percentage of the test compound readout over those of negative controls at a concentration of 10 uM.
Ratio(cpd): Ratio of the test compound.
Ratio(Blank): Ratio of the blank control without stimulus buffer.
Ratio(NC): Ratio of the negative controls with stimulus buffer.
17. Outcome assignment:
If the compound causes 3SD (of negative controls) or more Percentage (%) decrease (of negative controls) AND the fluorescence readout of the compound is within 3SD of the fluorescence of the negative controls, the compound is considered to be active.
If the compound is active in both duplicates and active in the primary screen, the compound is confirmed as active as an inhibitor/blocker of the Kir2.1 channels in the Outcome. Otherwise, it is designated as inactive.
18. Score assignment:
An inactive test compound is assigned the score of 0.
An active test compound is assigned a score between 0 and 100 by calculation of Integer(Ln(Abs(100- avPercentage)+1)*20), where avPercentage is the average of the duplicates of the test compound.
List of reagents
1. Kir2.1 HEK293 cell lines (provided by JHICC)
2. PBS: pH7.4 (Gibco, Cat#10010)
3. Medium: DMEM/F12 50/50 (Mediatech, Cat#15-090-CV)
4. Fetal Bovine Serum (Gemini, Cat# 100-106)
5. 200 mM L-Glutamine (Gibco, Cat#25030)
6. 100x Penicillin-Streptomycin (Mediatech, Cat#30-001-CI)
7. 0.05% Trypsin-EDTA (Gibco, Cat#25300)
8. G418 (Geneticin): (Gibco, Cat#11811-031)
9. HEPES (Sigma, Cat#H4034)
10. Chlorpromazine hydrochloride (Sigma, C8138)
11. FluxOR detection kit (Invitrogen, Cat #F10017): FluxOR, assay buffer and stimulus buffer.
12. Triple-layer flask (VWR, Cat #62407-082)
13. BD Biocoat 384-well plates (BD, Cat# (35)6663 and Lot #8163495)
14. 10x HBSS (Gibco, Cat#14065)
Possible artifacts of this assay can include, but are not limited to: non-intended chemicals, or dust in or on wells of the microtiter plate, compounds that non-specifically modulate the cell host or the targeted activity, compounds that induce K/Tl flux independent of Kir2.1, compounds that directly interact with the Tl sensitive dye molecules, and compounds that quench or emit light or fluorescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary, based upon the actual sample provided by the MLSMR.
1: avPercentage of Tested compounds (10uM**) Average percentage of the duplicates of the test compound readout (Percentage (%)) at a concentration of 10 uM.
** Test Concentration.
Data Table (Concise)