Specificity screen against TRPC6 for compounds that inhibit transient receptor potential cation channel C4 (TRPC4)
Assay Implementation: Melissa Miller, Amy Scott, Shunyou Long M.S., Meng Wu Ph.D., Joseph Babcock, Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D. ..more
BioActive Compounds: 108
Data Source: Johns Hopkins Ion Channel Center
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: Mike Zhu Ph.D., Ohio State University
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R21 NS056942-01
Grant Proposal PI: Mike Zhu Ph.D.
Assay Implementation: Melissa Miller, Amy Scott, Shunyou Long M.S., Meng Wu Ph.D., Joseph Babcock, Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D.
Name: Specificity screen against TRPC6 for compounds that inhibit transient receptor potential cation channel C4 (TRPC4)
See the related assay (Pubchem AID: 2247)
The purpose of this assay is to assess the specificity of those compounds identified as inhibitors of the transient receptor potential cation channel C4 (TRPC4) from the primary screen (AID: 2247). This specific counter screen is to eliminate any compounds that interact with TRPC6, rather than TRPC4 mu-opioid receptor expressing HEK293 cells. The protocol as in AID 2553 is used for this assay. Compounds were tested in duplicate and their effects were evaluated by the calculated integrated fluorescence ratio percentage, normalized with positive controls. If the compound causes a decrease in fluorescence signal greater than the mean of control wells minus 3SD it is considered active. Those compounds active in both duplicates are considered inhibitors of membrane potential in the presence of TRPC6, and therefore are NOT specific to TRPC4 channels.
1. Cell culture: Cells are routinely cultured in DMEM/high glucose medium, supplemented with 10% Heat Inactivated Fetal Bovine Serum (HiFBS), 50 IU/ml penicillin, 50 ug/mL streptomycin, and 400 ug/mL G418
2. Cell plating: Add 50 ul/well of 300,000 cells/ml re-suspended in DMEM/high glucose medium with 10% HiFBS, 50 IU/ml penicillin, and 50 ug/mL streptomycin
3. Incubate overnight at 37C and 5% CO2
4. Remove medium and add 20 ul/well of Membrane Potential Dye
5. Incubate 45 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 (EC0), ECmax of Acetylcholine
7. Load cell plates to Hamamatsu FDSS 6000 kinetic imaging plate reader
8. Measure fluorescence for 5 seconds at 1Hz to establish baseline
9. Add 4ul of 7.5x compound stock into the cell plates.
10. Incubate plates for 110 seconds
11. Add 6ul maximally activating concentration of Acetylcholine (Acetylcholine ECmax) and read for another 110 seconds.
12. Calculate ratio readout as F(max-min)/F0 and integrated ratio readout
13. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z' factors
14. Outcome assignment: If the compound caused a decrease of the membrane potential signal greater than 3SD of the positive control and retained a positive fluorescent signal, it is termed active. If the compound is active in both duplicates and active in the primary screen (pubchem AID: 2247), the compound is considered a non-specific modulator of membrane potential in HEK293 cells expressing TRPC6 channels (Value=2). Otherwise, it is designated as inactive (Value=1).
15. Score assignment: An inactive test compound is assigned the score of 0. An active test compound is assigned a score between 5 and 100 by calculating Integer(24.3*log(AvPercentage - 0.27))
List of reagents
1. TRPC6-expressing HEK293 Cells (provided by Assay Provider Michael Zhu, Ohio State University)
2. Medium: Dulbecco's Modified Eagle Medium (D-MEM) (1X), liquid (high glucose) w/L-Glut (Sigma, Cat# D5796-500ML)
3. Heat Inactivated Fetal Bovine Serum (Sigma, Cat# F2442)
4. 100x Penicillin-Streptomycin (Mediatech, Cat# 30-001-CI)
5. CellStripper (Mediatech, Cat# 25-056-Cl)
6. G418: (Invitrogen, Cat# 11811-031)
7. HEPES (Sigma, Cat# H4034)
8. 10XHBSS (#Invitrogen Cat# 14065056)
9. Acetylcholine chloride (Sigma, Cat# A6625)
10. 2-APB (Sigma, Cat# D9754)
11. Membrane Potential Assay Kit, Blue (Molecular Devices, Cat# R8034)
12. Triple-layer flask (VWR, Cat# 62407-082)
13. BD Biocoat 384-well plates (BD, Cat# (35)4663 and Lot# 8281903)
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, 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.
** Test Concentration.
Data Table (Concise)