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BioAssay: AID 720647

7011-01_Antagonist_SinglePoint_HTS_Activity

Assay Overview: HEK293 cells are utilized to screen for LGR2 GPCR antagonists. First, cells are transfected with a construct to express the Bursicon agonist. Conditioned media is generated in bulk. Separately, HEK293 cells are co-transfected with plasmid encoding the cDNA for the wild type Bursicon receptor and a CRE-luciferase reporter gene. After 24 hours, cells are treated with compound for 30 minutes and then stimulated with Bursicon conditioned media at half maximal stimulation. After 4 hours, cells are lysed with Promega SteadyGlo to measure luciferase with a Perkin-Elmer ViewLux. ..more
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 Tested Compounds
 Tested Compounds
All(375643)
 
 
Active(1337)
 
 
Inactive(373538)
 
 
Inconclusive(788)
 
 
 Tested Substances
 Tested Substances
All(378311)
 
 
Active(1340)
 
 
Inactive(376183)
 
 
Inconclusive(788)
 
 
 Related BioAssays
 Related BioAssays
AID: 720647
Data Source: Broad Institute (Bursicon-induced LGR2 mediated cAMP production in LGR-2/CRE6..)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-10-17

Data Table ( Complete ):           Active    All
BioActive Compounds: 1337
Description:
Keywords: HEK293, LGR2, Bursicon


Assay Overview: HEK293 cells are utilized to screen for LGR2 GPCR antagonists. First, cells are transfected with a construct to express the Bursicon agonist. Conditioned media is generated in bulk. Separately, HEK293 cells are co-transfected with plasmid encoding the cDNA for the wild type Bursicon receptor and a CRE-luciferase reporter gene. After 24 hours, cells are treated with compound for 30 minutes and then stimulated with Bursicon conditioned media at half maximal stimulation. After 4 hours, cells are lysed with Promega SteadyGlo to measure luciferase with a Perkin-Elmer ViewLux.


Expected Outcome: Antagonists will lead to a decrease in luminescence signal.
Protocol
Protocol

Day 0:
Cell Culture
HEK 293 cells are plated in DMEM culture media (containing 10% Fetal Bovine Serum (FBS) and 1X penn/strep/glutamine) at a density of 5x106 cells/T175 flask and grown for 3 days at 37 degrees C in 5% CO2 incubator.

Day 3:
Transient Transfection
Cells are transiently transfected in DMEM transfection media (containing 1X penn/strep/glutamine without serum) using polyethyleneimine (PEI).
1)Mix 2.9ug/flask of cDNA encoding the wild-type Bursicon Receptor with 14.6ng/flask of cDNA encoding a CRE-luciferase reporter gene with a PEST/HCL sequence in 2mL of transfection media
2)Add 61uL/flask of PEI of 1mg/mL in 1.5mL of transfection media
3)Mix the 2mL cDNA solution with 1.5mL of PEI solution and incubate at RT for 20 mins
4)Aspirated the culture media from the cells in T175 flask
5)Add 25 mL of transfection media and 3.5mL of transfection mixture to T175 falsk.
6)Mix the media with transfection mixture well and transfect for 2 days at 37 degrees C in 5% CO2 incubator

Day 5:
Cell Plating
1)Typsinize the Cells with 5mL of 0.05% Trypsin and incubate at 37oC for 5 min.
2)Add 5mL of DMEM assay media (containing DMEM without phenol red, 10% NuSerum and 1x Pen/Strep/Glutamine) to inactive trypsin
3)Collect cells by centrifugation at 1000rpm for 5 min
4)Suspend the cells in DMEM assay media to a cell density of 4x105 cells/mL
5)Plate the cells to 1536-well Aurora Mako plate with 2000 cells/well/5uL using ViaFill
6)Incubate the cells at 37 degrees C in 5% CO2 incubator on GS system for overnight

Day 6:
Compound Treatment, Agonist Stimulation and Detection
Assay Setup
1)Thaw aliquoted Bursicon conditioned media and diluted with DMEM assay media at a 1/20 dilution. Filter the solution through a 0.22uM filter
2)Add DMEM condition media, the diluted Bursicon conditioned media and SteadyGlo to the bottles and setup BioRAPTR

Run automation protocol on GS system
1)Take the assay plate with transfected cells from the incubator
2)Transfer 5nL/well of 10mM compound to assay plate
3)Incubate 30 mins in incubator
4)Add 1uL/well of DMEM assay media to PosCon wells and diluted Bursicon solution to the other wells using BioRAPTR (Beckman) based on the plate map
5)Incubate the assay plate for 4 h in incubator
6)Take the plate out from the incubator and equilibrate the assay plate at RT for 10 mins
7)Add 1uL/well of SteadyGlo to assay plate using BioRAPTR, incubate at RT for 10 mins
8)Read the plate on ViewLux (PerkinElmer) for Luminescence.

Culture Media
DMEM high glucose, no glutamine (Invitrogen, 11960)
Pen/Strep/Glutamine (Invitrogen 10378-016)
Fetal Bovine Serum (Atlanta Biological, S10350)

Transfection Media
DMEM high glucose, no glutamine (Invitrogen, 11960)
Pen/Strep/Glutamine (Invitrogen 10378-016)

Assay Media
DMEM no phenol red (Invitrogen, 21063-029)
Pen/Strep/Glutamine (Invitrogen 10378-016)
NuSerum (BD biosciences, 24883)

0.05% Trypsin-EDTA (Invitrogen, 25300-062)
Aurora 1536-well Mako plate (Aurora/Brooks, 00028778)
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1REPRODUCIBILITY_COSINE_TRANSFORMA measure of how well the activity reproduced across the two samples. Computed as the absolute value of the cosine between the 'replicate vector' (ScoreA, ScoreB ---as well as ScoreC and/or ScoreD where applicable) and the vector (1, 1) representing perfect reproducibility. NULL will appear in this column if a sample was not run in duplicate or if the data produced by one of the replicates was InvalidFloat
2PCT_ACTIVE_REPLICATESThe percentage of replicates which pass the activity threshold.Float
3REPLICATE_A_ACTIVITY_SCORE_9.99uM_(%)The calculated activity for the indicated sampleFloat%
Additional Information
Grant Number: 1R03MH097534-01

Data Table (Concise)
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