AID	Name	Type	Comment
435007	Summary of Broad Institute MLPCN Beta Cell Apoptosis Inhibition Project	summary	Summary assay

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:
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)

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.


ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (HVC) was determined and the active concentration limit (AC_limit) was set to equal (10)(HVC).


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.


MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION(AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.


PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
b) AC > AC_limit, or
c) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null

Activity_Outcome = 2 (active) when:
AC <= AC_limit

Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= 30% but <= 70%, or
b) The fit was not valid due to poor fit quality.


PUBCHEM_ACTIVITY_SCORE:

PUBCHEM_ACTIVITY_SCORE = 0 when PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive)

PUBCHEM_ACTIVITY_SCORE = (-10)[Log(AC)], where AC is in molar, when PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
Scores relate to AC in this manner:

120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M

PUBCHEM_ACTIVITY_SCORE = 100 when the curve fit is constant and showing full inhibition at all tested concentrations.


Note:

The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.

All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.

Grant Number: DP2 DK083048