Confirmation dose response assay for compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1
Assay Implementation: Meng Wu Ph.D., Shunyou Long M.S., Haibo Yu Ph.D., Hao-ran Wang Ph.D., Bill Shi Ph.D., David Meyers Ph.D., and Jia Xu Ph.D. ..more
BioActive Compounds: 22
Depositor Specified Assays
Data Source: Johns Hopkins Ion Channel Center (JHICC)
BioAssay Type: Confirmatory, Confirmatory Screening, Multiple 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., Shunyou Long M.S., Haibo Yu Ph.D., Hao-ran Wang Ph.D., Bill Shi Ph.D., David Meyers Ph.D., and Jia Xu Ph.D.
Name: Confirmation dose response assay 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 for the confirmation dose responses for compounds that inhibit/block inward-rectifying potassium ion channel Kir2.1, that identified in the primary screen assay (PubChem AID 1672) and consequent specificity screens (PubChem AID 1843). It employs the same experimental conditions as presented in the primary screen assay, except in multiple concentrations. Compounds were tested in triplicates and their effects were evaluated by the calculated FluxOR fluorescence ratio.
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. Outcome assignment:
If the test compound causes inhibition of the Kir2.1 in any concentrations tested AND a dose response is generated, the compound is considered to be active.
If the test compound does not cause inhibition of the Kir2.1 current in any concentrations tested OR a dose response is not generated, the compound is designated as inactive.
17. Score assignment:
An inactive test compound is assigned the score of 0.
An active test compound is assigned the score of 100.
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, 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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)