Counter 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: 426
Data Source: Johns Hopkins Ion Channel Center (JHICC_Kir2.1_Counter_1)
BioAssay Type: Confirmatory, Counter Screen, 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: Counter 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 counter screen the compounds that identified in the primary screen assay (PubChem AID 1672) against their non-specific effects on parental HEK293 cells of Kir2.1-HEK293 cells. It employs the same experimental conditions as presented in the primary screen assay, except the usage of parental HEK293 cells, instead of Kir2.1-HEK293 cells. Compounds were tested in duplicates and their effects were evaluated by the calculated FluxOR fluorescence ratio percentage normalized negative controls without compounds. If the compound causes 3SD (of negative controls) or more activity decrease OR increase in both duplicates, the compound is considered to be active that has non-specific effects on the parental HEK293 cells. It is NOT specific to inward-rectifying potassium ion channel Kir2.1.
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: Cells were re-suspended in DMEM/F12 medium with 10% FBS at 300,000 cells/ml and added to the 384 well microtiter plates at 50 ul/well
3. Incubate overnight at 37#C and 5% CO2
4. Remove medium and add 25 ul/well of 1x FluxOR solution
5. Incubate 90 minutes at room temperature (RT) in the dark
6. Prepare 7.5X compound plates and control plates on 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
8. Add 4 ul of 7.5x compound stock into the cell plates using 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 OR increase 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, the compound is confirmed as active as a non-specific modulator for the parental HEK293 cells 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 greater than 0 by calculating Integer(Ln(Abs(100- avPercentage)+1)*20), where avPercentage is the average of the duplicates of the test compound.
List of reagents
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, dust in or on wells of the microtiter plate, or 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. The condition is optimal for screening for compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1, NOT for the assay of HEK293 modulators. Normalization is to this set of data and cannot be used for comparison with other counter screens.
** Test Concentration.
Data Table (Concise)