Cell Viability - NIH 3T3
We have developed a 1536-well cell-based assay for quantitative high throughput screening (qHTS) against a number of cell lines to determine in vitro cytotoxicity of small molecules. This particular assay uses the NIH 3T3 fibroblast cell line which is established from NIH Swiss mouse embryo. ..more
BioActive Compounds: 171
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]
National Institutes of Environmental Health Sciences [NIEHS]
National Toxicology Program [NTP]
NCGC Assay Overview:
We have developed a 1536-well cell-based assay for quantitative high throughput screening (qHTS) against a number of cell lines to determine in vitro cytotoxicity of small molecules. This particular assay uses the NIH 3T3 fibroblast cell line which is established from NIH Swiss mouse embryo.
NCGC Assay Protocol Summary:
The CellTiter-Glo luminescent cell viability assay (Promega) is a homogeneous method to measure the number of viable cells in culture. The end point readout of this assay is based on quantitation of intracellular ATP, an indicator of metabolic activity, using the luciferase reaction. Luciferase catalyzes the oxidation of beetle Luciferin to oxyluciferin and light in the presence of ATP. The luminescent signal is proportional to amount of ATP present.
Using the CellTiter-Glo luminescent cell viability assay, the amount of cellular ATP was measured in the NIH 3T3 cell line with complete culture medium following compound treatment for 40 hours. The assay was performed in opaque white Kalypsys 1536-well plates. In the screen, tamoxifen and doxorubicin were used as positive controls. Library compounds were measured for their ability to cause acute toxicity in the cell line, as reflected by a decrease in intracellular ATP levels, in a concentration-dependent manner. Data were normalized to the controls for basal activity (DMSO only) and 100% inhibition (100 uM tamoxifen). AC50 values were determined from concentration-response data modeled with the standard Hill equation.
qHTS protocol for CellTiter Glo NIH 3T3 cellular assay
[Step] [Parameter] [Value] [Description]
1. Reagent; 5 uL; 1000 NIH 3T3 cells/well
2. Time; 5 hr; 37 oC incubation
3. Compounds; 23 nL; 0.59 nM to 92 uM
4. Controls; 23 nL; Tamoxifen 0.34 uM to 100 uM & Doxorubicin 0.02 nM to 100 uM
5. Time; 40 hr; 37 oC incubation
6. Reagent; 5 uL; CellTiter Glo reagent
7. Time; 20 min; Room Temperature
8. Detection; Luminescence; Viewlux plate reader
Keywords: cell viability, cytotoxicity, NIH 3T3 cell line, luminescence, NTPHTS, NTPHTS_NCGC, NIEHS, EPA, DSSTox, MLSMR, MLSCN, NIH Roadmap, qHTS, NCGC
1. Compounds are first classified based on type of titration curve, and quality of curve fit and efficacy as being active (probable cytotoxic; complete curve and good quality fit, or partial curve, good quality fit and high efficacy), inactive (flat curve), or inconclusive (other).
2. Within the active (cytotoxic) compounds, compounds were ranked by efficacy and potency. Compounds with the highest combined efficacy and potency are assigned a score of 100 and lowest a score of 50. Inactive compounds are assigned a score of 0, and inconclusive compounds are assigned a score of 25.
Data Table (Concise)