Statistics of Assay Validation in High Throughput Cell Imaging of Nuclear Factor κB Nuclear Translocation

This report describes statistical validation methods implemented on assay data for inhibition of subcellular redistribution of nuclear factor κB (NF κB) in HeLa cells. We quantified cellular inhibition of cytoplasmic–nuclear translocation of NF κB in response to a range of concentrations of interleukin-1 (IL-1) receptor antagonist in the presence of IL-1α using eight replicate rows in each four 96-well plates scanned five times on each of 2 days. Translocation was measured as the fractional localized intensity of the nucleus (FLIN), an implementation of our more general fractional localized intensity of the compartments (FLIC), which analyzes whole compartments in the context of the entire cell. The NF κB antagonist assay (inhibition of IL-1- induced NF κB translocation) data were collected on a Q3DM (San Diego, CA) EIDAQ™ 100 high throughput microscopy system. [In 2003, Q3DM was purchased by Beckman Coulter Inc. (Fullerton, CA), which released the IC 100 successor to the EIDAQ 100.] The generalized FLIC ...