Luminescence Cell-Based Dose Retest to Confirm Inhibitors of Beta Cell Apoptosis
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 ..
BioActive Compounds: 2159
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 afterward. 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.
Cytokine induced apoptosis leads to cell death and thus, reduced ATP levels. Candidate compounds will prevent cell death and keep ATP levels elevated. ATP is measured with Cell Titer go (Promega) and so increased values are expected. A compound is scored as a hit when an increase in cellular ATP occurs with an IC50 of 10 uM or less.
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 37C 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 8 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
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)
Dose Data Analysis
Neutral control wells (NC) and positive control wells (PC) were included on every plate.
Active compounds result in increased readout signal.
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 NC wells was set to a normalized activity value of 0.
The median raw signal of the intraplate PC wells was set to a normalized activity value of 100.
Experimental wells were scaled to this range, giving an activity score as percent change in signal relative to the intraplate controls.
No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
AC50 values were calculated using the curve fitting strategies in Genedata
Screener Condoseo (7.0.3). AC50 values were extrapolated up to 1 log
over the highest tested concentration.
PubChem Activity Score and Outcome
Inactive compounds = 0
Active compounds = -10*Log(AC50)
Superactive compounds (full inhibition at all concentrations; constant curve fit) = 100
Activity_Outcome = 1 (inactive)
AC50 > 1 log over the highest tested concentration
Activity_Outcome = 2 (active)
AC50 <= 1 log over the highest tested concentration
Activity_Outcome = 3 (inconclusive)
Curve fitting strategy resulted in a constant fit with activity >30% but <70%
The fit was not valid due to poor fit quality.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)