NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Screening Centers Network [MLSCN]
National Institutes of Environmental Health Sciences [NIEHS]
National Toxicology Program [NTP]
Grant number: None

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 caspase 3/7 of small molecules. This particular assay uses the Jurkat cell line which is derived from human T cell leukemia.

NCGC Assay Protocol Summary:

The Caspase-Glo 3/7 assay (Promega) is a homogeneous method to measure caspase-3 and caspase-7 activities in cultured cells. The quantitation of caspase 3/7 activity is based on the cleavage of a peptide-aminoluciferin substrate. After caspase cleaves the substrate the free aminoluciferin is liberated and serves as the substrate of luciferase, which generates the light. The luminescent signal is proportional to amount of caspase 3/7 activity present.

Using the Caspase-Glo 3/7 assay, the amount of caspase 3/7 activity was measured in the Jurkat cell line with complete culture medium following compound treatment for 16 hours. The assay was performed in opaque white 1536-well plates. In the screen, tamoxifen and doxorubicin were used as positive controls. Library compounds were measured for their ability to cause the activation of caspase 3/7 in the cell line, as reflected by an increase in luminescence signal, in a concentration-dependent manner. Data were normalized to the controls for basal activity (DMSO only) and 100% stimulation (100 uM tamoxifen). AC50 values were determined from concentration-response data modeled with the standard Hill equation.

qHTS protocol for caspase 3/7 assay in Jurkat cells
[Step] [Parameter] [Value] [Description]
1. Reagent; 5 uL; 5000 Jurkat cells/well
2. Compounds; 23 nL; 0.59 nM to 92 uM
3. Controls; 23 nL; Tamoxifen 0.23 uM to 100 uM & Doxorubicin 7.0 pM to 100 uM
4. Time; 16 hr; 37#C incubation
5. Reagent; 5 uL; Caspase-Glo 3/7 reagent
6. Time; 20 min; Room Temperature
7. Detection; Luminescence; Viewlux plate reader

Keywords: caspase 3/7, apoptosis, Jurkat cell line, luminescence, NTPHTS, NTPHTS_NCGC, NIEHS, EPA, DSSTox, MLSMR, MLSCN, NIH Roadmap, qHTS, NCGC

Compound Ranking:

1. Compounds are first classified based on type of titration curve, and quality of curve fit and efficacy as being active (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 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. Less conclusive activators are assigned a score of 40, likely activators are assigned a score of 30, inconclusive compounds are assigned a score of 15, and inactive compounds are assigned a score of 0.