Fluorescent Imaging of Extracellular PIA in S. aureus

For fluorescent imaging of extracellular PIA, S. aureus cells were stained using WGA-Alexa 488 (Invitrogen, Carlsbad, CA, USA) according to a previously published method (28 (link)), with modifications. An overnight culture of S. aureus SH1000 was diluted 500-fold in BHIG broth containing 5% DMSO, with or without test compounds (20 μM JBD1, 20 μM ANG1, 20 μM JBD1 and 100 μM MK-7, or 100 μM MK-7), and 2-mL aliquots were cultured in 12-well flat-bottomed polystyrene plates (Corning) at 37°C for 24 h under static conditions. After removing the supernatants, the sedimented cells were resuspended in PBS, spotted on glass slides, and left to dry at room temperature. Then, the cells were stained with 5 μg/mL WGA-Alexa 488 (Invitrogen) in PBS for 20 min at 37°C, washed twice with PBS, and subsequently stained with DAPI (Wako, Osaka, Japan) for 5 min at room temperature. Stained cells were mounted with the Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) and observed using a fluorescence microscope (ECLIPSE E600; Nikon, Tokyo, Japan).