12 well flat bottomed polystyrene plates

An overnight culture of S. aureus SH1000 was diluted 500-fold into BHIG broth 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 4 or 24 h under static conditions. Cells from 1 mL of the culture were harvested by centrifugation at 5,000 × g and 25°C for 10 min and resuspended in 1 mL of PBS. Then, 1 μL of component A (preliminarily diluted 10-fold with DMSO) included in the BacLight RedoxSensor Green Vitality Kit (Invitrogen) was added to the cells and incubated for 10 min at room temperature. Cells were collected by centrifugation, fixed in 4% PFA for 5 min at room temperature, washed twice, and resuspended in 1 mL of PBS. The cell suspensions were diluted 10-fold with DDW, and stained cells were analyzed using a flow cytometer (CytoFLEX, Beckman Coulter, CA, USA) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Cells were flowed at 10 μL/min, and 10,000 events were recorded for each sample.

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).