Luminescence Cell-Based Secondary HTS screen to identify cytotoxic compounds of NIH3T3 cells
Assay Overview: The assay detects compounds that are cytotoxic to NIH3T3 cells after approx. 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: 3
Keywords: NIH3T3, cytotoxic
Assay Overview: The assay detects compounds that are cytotoxic to NIH3T3 cells after approx. 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)
B. Reagents, materials 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#C. Warm up to 37#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#C. Warm up to 37#C in water bath before use.
C. 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.
-Rinse cells with 10 ml PBS/plate.
-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
D. 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).
Dose Data Analysis
Neutral control (NC) wells and positive control (PC) wells were included on every plate.
Active inhibitor compounds result in decreased signal.
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 NC wells was set to a normalized activity value of 0.
2X the median raw signal of the intraplate NC wells was set to a normalized activity value of +100.
0.5X the median raw signal of the intraplate NC wells was set to a normalized activity value of -50.
Experimental wells were scaled to this range, giving an activity score as percent change in signal relative to the neutral controls.
No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
IC50 values were calculated using the curve fitting strategies in Genedata
Screener Condoseo (7.0.3). IC50 values were extrapolated up to 1 log
over the highest tested concentration.
PubChem Activity Score and Outcome
Inactive compounds = 0
Active compounds = -10*Log(IC50)
Activity_Outcome = 1 (inactive)
IC50 > 1 log over the highest tested concentration
Activity_Outcome = 2 (active)
IC50 <= 1 log over the highest tested concentration
Activity_Outcome = 3 (inconclusive)
Curve fitting strategy resulted in a constant fit with activity >30% but <70%
The fit was not valid due to poor fit quality.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)