NIH/3T3(mouse embryonic fibroblast cell line) toxicity Measured in Cell-Based System Using Plate Reader - 2017-02_Inhibitor_Dose_DryPowder_Activity_Set2
Assay Overview: The assay 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
BioActive Compounds: 22
Keywords: NIH3T3, cytotoxic
Assay Overview: The assay 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 determine compounds that might yield false positive by killing host cell in T. Cruzi invasion assays.
PROTOCOL HTS ASSAY in 384-well plate
A. Cells and parasite:
- NIH/3T3 cells (mouse embryonic fibroblast cell line)
C. Reagents, materials, solutions and culture media:
- 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
- T175, T225 culture flasks with vented caps (BD Falcon, cat number: 353028)
- Corning Hyperflasks (Corning, cat number: 10024)
- For cell propagation:
90% DMEM+Phenol red, 10% FBS, 1% PSG. Mix and filter through 0.2 microns membrane. Keep at 4 degrees C. Warm up to 37 degrees C 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 4 degrees C. Warm up to 37 degrees C in water bath before use.
D. cell culture
- 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.
- 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, pipet 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
E. Growth inhibition assay for HTS in 384 well plates:
- 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.
- 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 ultrasensitive luminescence program on the Envision (Perkin Elmer).
PRESENCE OF CONTROLS: Neutral control wells (NC; n=132) and positive control wells (PC; n=18) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:
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 (10)(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): 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.
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.
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:
1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.
2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.
3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.
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 Format: Cell-based
Assay Type: Toxicity
Assay Cell Type: NIH 3T3
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)