Mammalian cell toxicity counterscreen to identify toxic HSP40 inhibitor compounds in NIH3T3 cells Measured in Cell-Based System Using Plate Reader - 2120-03_Inhibitor_Dose_CherryPick_Activity_Set2
Retest at dose using human epithelial adenocarcinoma NIH3T3 cells to identify small molecules toxic to living cells. The toxicity of the small molecules will be measured by the conversion of luciferin to oxyluciferin and light (luminescence) mediated by the luciferase and the ATP present in cells. Toxic compounds will diminish intracellular ATP level in the cells and this will result in a decrease of luminescent signal. ..more
BioActive Compounds: 44
Keywords: Malaria, cell toxicity, Heat shock protein
Luciferase assay (Celltiter-Glo, Promega).
Retest at dose using human epithelial adenocarcinoma NIH3T3 cells to identify small molecules toxic to living cells. The toxicity of the small molecules will be measured by the conversion of luciferin to oxyluciferin and light (luminescence) mediated by the luciferase and the ATP present in cells. Toxic compounds will diminish intracellular ATP level in the cells and this will result in a decrease of luminescent signal.
Expected Outcome: Compounds with an AbsAC-35 below 20 uM will be considered toxic and will be discarded from further analysis. Active compounds with an AbsAC-35 above 20 uM will not be considered toxic and will continue on to further analyses.
Protocol: Cell toxicity assay (NIH3T3 cells)
Mouse fibroblast NIH3T3 cell line (ATCC, CR-1658) is propagated in DMEM media without phenol red (Invitrogen, 31053) supplemented with 10% fetal bovine serum (Gibco, 26140-079), 1% penicillin/streptomycin/glutamine (Invitrogen, 10378-016)) and sodium pyruvate 1mM (Gibco, 11360) at 37degrees C in CO2 incubators (Thermo Scientific) with 5% CO2, 21% O2, and 95% humidity. Cells are grown in T175 flask (BD Falcon, Ref# 353112) harvested at more than 80% confluence using 7 ml Trypsin-EDTA 0.25% (Cellgro, Cat# 25-053-CL) for 5 minutes and then the trypsin is inactivated with 7 ml of complete medium respectively. Cells are centrifuged at 1000rpm/5min and resuspended in fresh complete DMEM media (Invitrogen, 31053) with 10% Heat inactivated fetal bovine serum (Invitrogen, Cat# 16140089), 1% penicillin/streptomycin/glutamine (Invitrogen, 10378-016) for compound screening. Cell number is counted using a Nexelcom Bioscience cell counter (Cellometer Auto M10).
Compound Screening is carried out on the Broad Institute/Chemical Biology Platform Walkup unit:
Day 1 (Cell plating):
1. NIH3T3 cells are harvested and resuspended in DMEM without phenol red (Gibco, 31053) with 10% Heat inactivated fetal bovine serum (Gibco, 26140-079) and 1% penicillin/streptomycin/glutamine (Invitrogen, Cat#10378-016) and sodium pyruvate 1mM (Gibco, 11360) . NIH3T3 cells (from an initial cell suspension of 125,000 cells/ml) are dispensed using a MultiDrop Combi/Standard tube dispensing cassette (Thermo Scientific) in white bottom 384 well assay plates (Corning, Cat.No. 8867BC) at a final density of 5,000 cells per well in final volume of 40 uL. The cells are kept in suspension using a magnetic bar and a stirrer during the dispensing.
2. The assay plate (cell plate) are placed in Cytomat Instruments cassettes (22 plates/cassette) and incubated for 24 hours at 37 degrees C in the Cytomat CO2 incubator calibrated at 5% CO2, 21% O2, and 95% humidity.
Day 2 (Compound pinning into assay plate):
3. The dose response compound plate (8 concentrations, 3 fold dilution, starting concentration 10 mM) are pinned twice using 384 well pin tool (100 nl) on pin table (Walkup Cybi Well) and transferred to assay plate. Pins are washed with methanol and DMSO between each pinning.
4. The assay plates treated with compounds are moving back to Cytomat CO2 incubator to be incubated for an additional 48 hours.
Day 4 (Reading luminescence from assay plates with Envision):
5. Each assay plate is pulled out of the incubator and cooled down at room temperature for 30 minutes. 30 uL/well (384 well) of Celltiter-Glo luciferase reagent 0.5X (diluted in H2O) (Promega, G7573) is dispensed using the the MultiDrop Combi dispensing cassette from Thermo Scientific. The assay plate returned to room temperature for 30 minutes to allow a complete cellular lysis.
6. Luminescence is measured (0.2 second/well) using luminescence detector (384-well) in each well using the Envision plate reader (Perkin Elmer)(Corning plate setting).
PRESENCE OF CONTROLS: Neutral control wells (NC; n=72) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): absACnn, the concentration at which the curve crosses threshold -35.0
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.
pAC was set to equal -1*log10(AC)
Activity_Outcome = 1 (inactive) when:
a) 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
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
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.
Categorized Comment - additional comments and annotations
Assay Type: Toxicity
Assay Format: Cell-based
Assay Type: Functional
Assay Cell Type: NIH3T3
* Activity Concentration. ** Test Concentration.
Data Table (Concise)