Second counter screen for compounds that inhibit transient receptor potential cation channel C4 (TRPC4)
Assay Implementation: Melissa Miller, Amy Scott, Shunyou Long M.S., Joseph Babcock, Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D., Meng Wu Ph.D. ..more
BioActive Compounds: 541
Data Source: Johns Hopkins Ion Channel Center
BioAssay Type: Second 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: Michael Zhu, Ph.D., Ohio State University
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R21 NS056942-01
Grant Proposal PI: Michael Zhu, Ph.D., Ohio State University
Assay Implementation: Melissa Miller, Amy Scott, Shunyou Long M.S., Joseph Babcock, Bill Shi Ph.D., David Meyers Ph.D., Jia Xu Ph.D., Meng Wu Ph.D.
Name: Second counter screen 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 counter screen those compounds that show inhibition of TRPC4 as identified in the primary screen (AID: 2247). This specific counter screen is to eliminate any compound that interacts with the mu-opioid receptor, rather than TRPC4. To do this, TRPC4 and the serotonin receptor (5HT1a) were stably expressed in a HEK293 cell line and channel activity monitored by calcium flux with a commercial Fluo4 kit. Compounds were tested in duplicate and were evaluated by calculation of the integrated fluorescence ratio and comparison to control wells. If the compound caused a decrease of the fluorescence signal greater than the mean normalized ratio of the ECMax control plus 3SD it is termed active. Those compounds active in both duplicate plates are considered inhibitors of calcium flux in this cell line. We expect that those compounds that show activity in both the TRPC4 mu-opioid receptor primary assay (AID: 2247) and this TRPC4-5HT1a assay are inhibitors of 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, 500 ug/mL G418 and 40 ug/mL hygromycin
2. Cell plating: Add 50 ul/well of 300,000 cells/ml re-suspended in DMEM/high glucose medium with 10% HiFBS.
3. Incubate overnight at 37C and 5% CO2
4. Remove medium and add 20 ul/well of 1x Fluo4 solution to cells
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), and ECmax of serotonin
7. Remove Fluo4 dye solution and add 40 ul/well of assay buffer to cells
8. Remove 40 ul solution and add 20 ul/well of assay buffer to cells
9. Load cell plates to Hamamatsu FDSS 6000 kinetic imaging plate reader
10. Measure fluorescence for 5 seconds at 1Hz to establish baseline
11. Add 4 ul of 7.5x compound stock into the cell plates.
12. Incubate plates for 110 seconds
13. Add submaximal concentration of serotonin and incubate for 110 seconds
14. Add maximally activating concentration of serotonin ( serotonin ECmax) and read for another 110 seconds.
15. Calculate ratio readout as F(max-min)/F0 and integrated ratio readout
16. Calculate the average and standard deviation for negative and positive controls in each plate, as well as Z and Z' factors
17. Outcome assignment: If the compound causes a decrease of the normalized ratio greater than the mean normalized ratio of the ECMax control plus 3SD, the compound is considered active. If the compound is active in both duplicates and active in the primary screen, the compound is considered an inhibitor of TRPC4 channels. Otherwise, it is designated as inactive.
18. Score assignment: An active compound is assigned to a score between 5 and 100 by calculating Integer(25.0 * log(AvPercentage + 7.50)); while an inactive compound is assigned to a score of 0.
List of reagents
1. TRPC4 and serotonin receptor-expressing HEK293 Cells (provided by Assay Provider Michael Zhu, Ohio State University)
2. PBS: pH7.4 (Invitrogen Cat#10010023)
3. Medium: Dulbecco's Modified Eagle Medium (D-MEM) (1X), liquid (high glucose) w/L-Glut (Sigma D5796-500ML)
4. Heat Inactivated Fetal Bovine Serum (Sigma, Cat# F2442)
5. L-Glutamine (Invitrogen, Cat#25030081)
6. 100x Penicillin-Streptomycin (Mediatech, Cat#30-001-CI)
7. CellStripper (Mediatech 25-056-Cl)
8. G418: (Invitrogen, Cat# 11811-031)
9. Hygromycin#(Mediatech, Cat#30-240-CR)
10. HEPES (Sigma, Cat#H4034)
11. 10XHBSS (#Invitrogen Cat#14065056)
12. Pluronic F-127 (20% solution in DMSO) (Invitrogen Cat#P3000MP)
13. Serotonin hydrochloride (Sigma, Cat# H9523)
14. Fluo-4 Calcium Assay Kit (Invitrogen, Cat # F14202)
15. Triple-layer flask (VWR, Cat #62407-082)
16. BD Biocoat 384-well plates (BD, Cat# (35)4663 and Lot #7346273)
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)