AID	Name	Type	Comment
624280	Broad Institute Inhibition of T.cruzi replication in culture Inhibitor Probe Project	summary	Summary assay

Keywords: NIH3T3, cytotoxic

Assay Overview: The assay detects compounds that are cytotoxic to NIH3T3 cells afer ~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 primary 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
- NIH/3T3 cells (mouse embryonic fibroblast cell line; ATCC CRL-1658)
- 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 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.
Solutions:
-D. 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, 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. Toxicity assay for HTS in 384 well plates:
Dilute to NIH/3T3 cells to 5000/60 ul of 2% FBS/DMEM media.
Plate 60 ul/well using Thermo MultiDrop Combi liquid dispenser and a sterilized standard sized dispensing cassette adding at a fast speed in a tissue culture hood.
Incubate cells 4-16 hrs at 37 C, 5%CO2.
Pin 100 nl compounds and incubate for an additional 90hrs.
Plates are equilibrated to room temperature for 1 hr.
30ul of diluted CellTiter-Glo (diluted 1:3 in PBS) is added to the well and incubated for 12 minutes.
Plates are read using the ultrasensistive luminescence program on the Envision (Perkin Elmer) 0.1s/well.

PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' 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.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.


PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at -24 (Greater than 3 times the standard deviation of the neutral control wells).

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 55.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T

Grant Number: 1 R03 MH085673-01