Quantifying NF-κB Nuclear Translocation

Cells were grown in black 96-well glass bottom plates (Costar). Measurement of NF-κB translocation from the cytoplasm to the nucleus was performed as previously described [13] (link) using a Cellomics NF-κB Activation Kit^™ (Thermo Fisher Scientific, Pittsburgh, PA, USA) according to the manufacturer’s protocol. The nucleus was immunostained with Hoechst 33342, and NF-κB was detected with an NF-κB primary antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody. Microscopic images were captured with the MetaFluor Imaging System as described above. Image analysis was performed using NIH ImageJ software (v.3.91, http://rsbweb.nih.gov/ij/) as described by Noursadeghi et al. [21] (link). NF-κB nuclear translocation was determined as an increase in the nucleus∶cytoplasm ratio.