Visualizing NF-κB Activation in Macrophages

In separate experiments, THP-1 cells were plated at 12-well plates and treated with phorbol 12-myristate 13-acetate (PMA) (InvivoGen, San Diego, CA, USA) at a concentration of 100 ng for 24 h to differentiate them to Mφ. A subset of Mφ was challenged with 10 nM of Porphyromonas gingivalis (a major periodontal pathogen) LPS (InvivoGen, San Diego, CA, USA). After 24 h, cells were stained for RELA primary antibody (rabbit polyclonal affinity purified antibody) and Alexa-Fluor (AF) 568 (Abcam, Waltham, MA, USA) as the secondary antibody as described before.^{16 (link)} DAPI and AF-488 Phalloidin (Invitrogen, Waltham, MA, USA) were used to stain nuclei and actin, respectively. Samples were visualized using a Nikon A1 confocal microscope. Quantification of nucleolus versus cytoplasmic staining was performed using ImageJ using three biological replicates as described.^{17 (link)}