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BioAssay: AID 743287

Luminescent GLuc Reporter Gene Assay Primary HTS to Identify Small Molecule Activator of Glucose Dependent Insulin Secretion Measured in Cell-Based System Using Plate Reader - 7055-01_Activator_SinglePoint_HTS_Activity

Keywords: Insulin, glucose-dependent insulin secretion, type 2 diabetes, Gaussia Luciferase, reporter gene ..more
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 Tested Compounds
 Tested Compounds
All(371106)
 
 
Active(2287)
 
 
Inactive(368851)
 
 
 Tested Substances
 Tested Substances
All(374203)
 
 
Active(2293)
 
 
Inactive(371910)
 
 
 Related BioAssays
 Related BioAssays
AID: 743287
Data Source: Broad Institute (7055-01_Activator_SinglePoint_HTS_Activity)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2014-02-24

Data Table ( Complete ):           Active    All
BioActive Compounds: 2287
Depositor Specified Assays
AIDNameTypeComment
743290Broad Institute Activators of glucose dependent insulin secretion Activator Probe Projectsummary
Description:
Keywords: Insulin, glucose-dependent insulin secretion, type 2 diabetes, Gaussia Luciferase, reporter gene

Assay Overview: The assay uses a rat Beta Cell line (INS-1E) engineered to contain a Gaussia luciferase reporter. Beta-cell lines expressing this construct co-secrete luciferase and insulin in close correlation.



Expected Outcome: Compounds that cause an increase in insulin secretion result in an increase in luminescence. Active hits are determined as wells with luminesncent signal four standard deviations above the per-plate population of neutral control wells (approximately 40 % of the IBMX positive control.)
Protocol
Cells were received from Collaborator: INS-1E parental containing a GLuc reporter gene

Cells were cultured in media containing:
RPMI 1640
1 mM Sodium Pyruvate
2 mM L-glutamine
55 uM Beta Mercaptoethanol
10% FBS, heat inactivated

Starvation Buffer/Assay Buffer: KRB, 0.1 % BSA
138 mM NACl
5.4 mM KCl
2.6 mM MgCl2
2.6 mM CaCl2
5 mM NaHCO3
0.1 % BSA

Assay protocol:
Grow cells to 80-90% confluency in T175 cm2 flasks.
On day of assay remove media and rinse flask with PBS. Add 15 mL of KRB, 0.1%BSA and incubate 30 minute in 37 degree, 5%CO2 incubator.
Remove KRB, 0.1%BSA buffer and rinse with PBS 2 times.
Add 3.5 mL of TrypsinLE to the flask and incubate 37 degrees Celsius/5% CO2 for 2 minutes at or until cells start to detach. Immediately add 20 ml of KRB, 0.1%BSA, 10% FBS.
Harvest cells and spin at 1000 RPM for 5 minutes.
Aspirate supernatant and resuspend cell pellet in KRB, 0.1% BSA.
Pass cell suspension through 0.4 uM filter.
Dilute cells to a concentration of 125,000 cells/mL in KRB, 0.1% BSA.
Add glucose to 11.2 mM in small batches of cell suspension.
Using the ViaFill add 6 ul of cell suspension to each well of 1536 Assay Ready Plates (750 cells/well).
Incubate cells with compounds 2 to 3 hours at 37 degrees Celsius, 5% CO2.
Thirty minutes prior to substrate reagent dilute substrate, native coelenterazine, in Phenol Red Free DMEM to a concentration of 75 uM.
Using the BioRaptR add 2 ul/well of assay plates for a final concentration of 18 uM native coelenterazine.
Read luminescence immediately on ViewLux.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in increasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the ''Stimulators Minus Neutral Controls" method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of 100.
Experimental wells values were scaled to this range.

PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise Pattern (Multiplicative)' in Genedata (v10.0.2) was applied to the normalized plate data.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the minimum of the normalized and corrected sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at 40.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 1 (inactive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
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 Invalid# Float
2PCT_ACTIVE_REPLICATESThe percentage of replicates which pass the activity threshold.Float
3REPLICATE_A_ACTIVITY_SCORE_16.64uM_(%) (16.64μM**)The calculated activity for the indicated sampleFloat%

** Test Concentration.
Additional Information
Grant Number: 1 R03 DA035188-01

Data Table (Concise)
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