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BioAssay: AID 488910

Detect Cellular ATP-levels in INS-1E Cells Measured in Cell-Based System Using Plate Reader - 2061-01_Inhibitor_SinglePoint_HTS_Activity

Assay Overview: INS1E rat insulinoma cells are similar to pancreatic beta cells because they secrete insulin in response to glucose stimulation. In the dose titration version of the primary assay, INS1E cells are treated with 3 cytokines (Interferon-gamma, Tumor necrosis factor-alpha and Interleukin 1-beta) and compounds for 48 hours. The combination of cytokines leads to apoptotic cell death. more ..
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 Tested Compounds
 Tested Compounds
All(19828)
 
 
Active(26)
 
 
Inactive(19796)
 
 
Inconclusive(6)
 
 
 Tested Substances
 Tested Substances
All(19828)
 
 
Active(26)
 
 
Inactive(19796)
 
 
Inconclusive(6)
 
 
 Related BioAssays
 Related BioAssays
AID: 488910
Data Source: Broad Institute (2061-01_Inhibitor_SinglePoint_HTS_Activity)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-10-26
Hold-until Date: 2011-06-13
Modify Date: 2011-06-13

Data Table ( Complete ):           Active    All
BioActive Compounds: 26
Depositor Specified Assays
AIDNameTypeComment
435007Summary of Broad Institute MLPCN Beta Cell Apoptosis Inhibition ProjectsummarySummary assay
Description:
Keywords: cytokine-induced apoptosis, pancreatic beta cells, INS1E insulinoma cells, Type I diabetes

Assay Overview: INS1E rat insulinoma cells are similar to pancreatic beta cells because they secrete insulin in response to glucose stimulation. In the dose titration version of the primary assay, INS1E cells are treated with 3 cytokines (Interferon-gamma, Tumor necrosis factor-alpha and Interleukin 1-beta) and compounds for 48 hours. The combination of cytokines leads to apoptotic cell death. The level of cell death is inferred my measuring overall ATP levels with Promega's Cell Titer Glo. On day 0, cells were plated at 8000 per well in 30 uL phenol red free RPMI.. On day 1, cytokines were added in 10 uL of phenol red free RPMI media and compounds were added immediately afterwards. Cells were incubated for 48 hours with cytokines and compounds. On day 3, 20 uL of Cell Titer Glo was added per well and ATP levels were measured with the Perkin Elmer Envision plate reader with settings for standard luminescence.
Protocol
Protocol:
Day 0: Collect cells and generate single cell suspension by trypsinization and passing through sterile 40 micron cell strainer (Falcon). Seed 8,000 cells/well of INS-1E rat beta-cell line in 30 uL media using white, opaque, bar coded, 384-well Corning 8867 plates; incubate at 37 degrees C overnight.
Day 1: Add 10 uL medium with cytokine cocktail to each well using the Combi. Pin transfer compounds to plates right after the addition of cyokines with 100 nL head and transfer 100 nL compound. Positive control added by double pinning plates.
Day 3: Add 20 uL Cell titer-Glo reagent to plates.
Agitate gently for 15 seconds to maximize cell lysis. Incubate 10 minutes.
Use Envision to read plate luminescence with standard luminescence parameters.

Cell carrying media:
RPMI 1640, 10% heat-inactivated fetal calf serum, 1 mM sodium pyruvate, 50 uM 2-mercaptoethanol, 2 mM glutamine, 10 mM HEPES, 100 U/ml penicillin, and 100 ug/ml streptomycin

Plating media:
RPMI 1640 (phenol red free), 5% heat-inactivated fetal calf serum, 1 mM sodium pyruvate, 50 uM 2-mercaptoethanol, 2 mM glutamine, 10 mM HEPES, 100 U/ml penicillin, and 100 ug/ml streptomycin
Cytokines: 10 ng/mL IL-1 beta (R&D Systems, 501-RL), 25 ng/mL TNF-alpha (R&D Systems, 410-MT), 50 ng/mL IFN-gamma (R&D 485-MI)

INS-1E media recipe, per Liter

RPMI-1640 (w/L-Glu & Glucose): to 1L
10% Fetal bovine serum: 100 mL
1M HEPES: 10 mL
100 mM Na pyruvate: 10 mL
55 mM Beta-mercaptoethanol: 909 uL
100x Pen-Strep: 10 mL


INS-1E plating recipe, per Liter

RPMI-1640 (No Phenol Red): to 1L
5% Fetal bovine serum: 50 mL
1M HEPES: 10 mL
100 mM Na pyruvate: 10 mL
55 mM Beta-mercaptoethanol: 909 uL
100x Pen-Strep: 10 mL


Notes:

- Seed 10 to 15 million per T175 in 20 mL media

- Seed 110 million per Hyperflask in ~550 mL

- Fluid change every 3 days, confluent in about 6-8 days.

- Plate 8,000 cells per well in 30 uL plating media

- 10 uL of cytokine mix is added on day run of assay (900 mL mix for a full run)

- Cytokine mix:

333 uL IFN-gamma
115 uL TNF-alpha
71 uL IL1-Beta
900 mL plating media


- Cell Titer Glo per run (20 uL per well x 384 wells x 220 plates=1700 mL)
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 (v7.0.3):
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 (v7.0.3) was applied to the normalized plate data.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean 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 50.

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 75.

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 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
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
2REPLICATE_A_ACTIVITY_SCORE (10μM**)The calculated percent activity for the indicated sampleFloat%
3REPLICATE_B_ACTIVITY_SCORE (10μM**)The calculated percent activity for the indicated sampleFloat%
4REPLICATE_C_ACTIVITY_SCORE (10μM**)The calculated percent activity for the indicated sampleFloat%

** Test Concentration.
Additional Information
Grant Number: DP2 DK083048

Data Table (Concise)
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