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BioAssay: AID 2010

Luminescence Cell-Based Dose Response HTS Screen to Identify Cytotoxic Compounds of NIH3T3 cells.

Assay Overview: This counter screen detects compounds that are cytotoxic to NIH3T3 cells after ~90hrs in culture. After plating the NIH3T3 cells, compounds are added to the wells. After 90 hours culturing, all cells in the well are lysed and ATP is detected using Cell Titer Glo ..more
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 Tested Compounds
 Tested Compounds
All(4063)
 
 
Active(1011)
 
 
Inactive(2724)
 
 
Inconclusive(328)
 
 
 Tested Substances
 Tested Substances
All(4065)
 
 
Active(1012)
 
 
Inactive(2725)
 
 
Inconclusive(328)
 
 
 Related BioAssays
 Related BioAssays
AID: 2010
Data Source: Broad Institute (2017-02_INHIBITORS_DOSE-TITRATION_MLPCN-CHERRYPICK)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-10-22
Modify Date: 2009-11-02

Data Table ( Complete ):           Active    All
BioActive Compounds: 1011
Depositor Specified Assays
AIDNameTypeComment
1885Luminescence Cell-Based/Microorganism Primary HTS to Identify Inhibitors of T.Cruzi Replicationscreening
1968Broad Institute MLPCN T. Cruzi Inhibition Projectsummary
Description:
Keywords: NIH3T3, cytotoxic

Assay Overview: This counter screen detects compounds that are cytotoxic to NIH3T3 cells after ~90hrs in culture. After plating the NIH3T3 cells, compounds are added to the wells. After 90 hours culturing, all cells in the well are lysed and ATP is detected using Cell Titer Glo

Expected Outcome: Compounds significantly suppressing luminescence, and therefore cytotoxic to NIH3T3s will be resolved as hits. This is a counter screen to AID 1885 to determine compounds that might yield false positive by killing host cell in T. Cruzi invasion assays.
Protocol
PROTOCOL HTS ASSAY in 384-well plate

A. Cells and parasite:
-NIH/3T3 cells (mouse embryonic fibroblast cell line)

B. Reagents, materials, solutions and culture media:
Reagents:
-DMEM with Phenol Red, high glucose, with L-glutamine and Sodium pyruvate (Cellgro, cat number: 10-013-CM)
-PSG or Penicillin-streptomycin-L-glutamine (Gibco-Invitrogen, cat number: 10378-016)
-FBS-Heat inactivated fetal bovine serum (Gibco-invitrogen, cat number: 16140-089)
-0.25% Trypsin-EDTA 1X (Gibco-Invitrogen, cat number: 25200-072)
-Sterile PBS (Phosphate Buffer Saline) 1X
-Materials:
-T175, T225 culture flasks with vented caps (BD Falcon, cat number: 353028)
-Corning Hyperflasks (Corning, cat number: 10024)
Culture medium:
-For cell propagation:
90% DMEM+Phenol red, 10% FBS, 1% PSG. Mix and filter through 0.2 microns membrane. Keep at 4c. Warm up to 37c in water bath before use.
-For Tc culture and assays:
98% DMEM+Phenol red, 2% FBS, 1% PSG. Mix and filter through 0.2 microns membrane. Keep at 4c. Warm up to 37c in water bath before use.
Solutions:

C. Cell culture NIH/3T3
Notes:
-NIH/3T3 cells are cultivated in DMEM supplemented with 10% FBS and 1% PSG in T175 or Corning Hyperflasks .
-Conditions can be adapted to other culture plate sizes.
Protocol:
-Aspirate medium.
-Rinse cells with 10 ml PBS/plate.
-Aspirate PBS.
-Add 4 ml of pre-warmed trypsin-EDTA, swirl the dish to make sure the trypsin covers all the cells.
-Leave the dish a few minutes at room temperature (usually not more than 5).
-Check that the cells are detaching.
-Add 21 ml of medium, pipette up and down to detach all the cells.
-Take 75 ul of media and add to Cellometer cassette (Nexcelom Biosciences). Using the Cellometer Auto T4 (Nexcelom Biosciences), focus so cell bodies have dark edges and clear/white centers. Select NIH3T3 preset menu, dilution 1x, display image, and count. Using this number dilute in either 2% FBS/DMEM for assays or 10% FBS/DMEM for propagation

D. Growth inhibition assay for HTS in 384 well plates:
Notes:
-Final volume per well for cells+compound is 50 uL.
-Final volume after adding substrate is ~65 ml. (evaporation of 50ul to 35 uL after 90 hrs, and then 30ul Cell Titer Glo)
-The assay is performed in DMEM+ 2% FBS and 1% PSG .
-Experiment set up is usually done ~12pm. Assay has to be incubated 90 hrs or a bit more.
Protocol:
-Warm up medium 2% FBS/DMEM
-Trypsinize NIH/3T3 cells as described in cell culture protocol
-When NIH3T3 are detached, harvest them in DMEM- 2% FBS and 1% PSG and count using the NIH3T3 program using Cellometer Auto T4 (Nexcelom Biosciences).
-Dilute cells to 166,667 cells /ml and add to flask.
-Plate 5,000 cells/50ul per well using a Thermo MultiDrop Combi liquid dispenser and a sterilized standard sized dispensing cassette adding at a fast speed in a tissue culture hood.
-Put back in incubator for 3 hours to allow cells to attach.
-Pin 50 nL compounds/DMSO to each well
-Incubate for 4 days (or minimum 90 hours).
-On day of substrate addition, prepare CELL TITER GLO
-Add 30 uL per well of 384 well plate using a Thermo MultiDrop Combi liquid dispenser and a sterilized standard sized dispensing cassette adding at a fast speed.
-Incubate for 10 minutes
-Read using the ultrasensistive luminescence program on the Envision (Perkin Elmer).
Comment
Data Analysis:

Normalization of all wells was performed using the 'Neutral Controls' one-point normalization method in Genedata Assay Analyzer, where the median of the neutral control well signals is set to normalized value of 0, and relative inhibition values of compound wells are set to the percentage of the 0 signal value. Plate pattern correction algorithms were not used.

IC50 values were calculated using the curve fitting algorithms of Genedata
Screener Condoseo (v7.0.3). EC50 values were extrapolated up to 1 log
over the highest tested concentration.

PUBCHEM_ACTIVITY_SCORE
Inactive compounds = 0
Active compounds = -10*Log(EC50)

PUBCHEM_ACTIVITY_OUTCOME
Activity_Outcome = 1 (inactive)
EC50 > 1 log over the highest tested concentration

Activity_Outcome = 2 (active)
EC50 <= 1 log over the highest tested concentration

Activity_Outcome = 3 (inconclusive)
Curve fitting strategy resulted in a constant fit with activity >30% but <70%
or
The fit was not valid due to poor fit quality.
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50 Qualifier'>', '=', or '<'String
2EC50*the concentration whereupon perceived activity reaches 50% of the maximumFloatμM
3EC50 Standard Errorthe standard error for the calculated EC50 valueFloatμM
4S0the fitted activity level at zero concentrationFloat%
5SInfthe fitted activity level at infinite concentrationFloat%
6Hill Slopethe slope at EC50Float
7Num. Pointsthe number of data points included in the plotInteger
8Max. Activitythe maximum activity value observedFloat%
9Activity at 0.10uM (0.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
10Activity at 0.19uM (0.19μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
11Activity at 0.38uM (0.38μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity at 0.75uM (0.75μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity at 0.80uM (0.8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity at 1.50uM (1.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity at 1.60uM (1.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity at 3.00uM (3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
17Activity at 6.00uM (6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH085673-01

Data Table (Concise)
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