Af 488 phalloidin

Subcellular distribution of proteins was determined by observation of cellular shapes and trajectories using phase-contrast microscopy followed by immediate fixation with 4% formaldehyde in 0.32 M sucrose in PBS for 15 minutes, permeabilization with 0.5% triton-X 100 for 10 minutes, blocking with PBS-BT (3% bovine serum albumin, 0.1% triton X-100, and 0.02% sodium azide in PBS) and staining in PBS-BT. Filamentous actin was stained with a 1:1000 dilution of 6.6 μM AF-488 Phalloidin (Invitrogen). Myosin distribution was stained with 1:200 polyclonal rabbit anti-myosin antibodies (ab2480, Abcam, Cambridge MA) in cells that were initially permeabilized and stabilized with a salt solution (50 mM imidazole, 50 mM KCl, 0.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA) containing 1% Triton-X 100, 4% PEG and 0.5 μM TMR-Phalloidin (Invitrogen); cells were then fixed in 4% formaldehyde in PBS, blocked with PBS-BT and stained with 1:1000 goat-anti-rabbit AF-488 (Abcam). Myosin was additionally visualized in living cells, 24 hours after transfection by electroporation with a plasmid containing a Xenopus myosin regulatory light chain-EYFP fusion transgene (gift of Aaron Straight).

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)}