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
BioActive Compounds: 2287
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.)
Cells were received from Collaborator: INS-1E parental containing a GLuc reporter gene
Cells were cultured in media containing:
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
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.
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.
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.
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.
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
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)