Quantification of Staphylococcus aureus Signaling Molecules

Overnight cultures of S. aureus 1:200 were grown in TSBG at 37°C with or without 50 μM clemastine, and samples were prepared for analysis as described for c-di-GMP with slight modifications (10 (link)). Briefly, the equal weights cell pellets were washed twice with cold molecular grade water (Corning) and lysed by homogenized for 5 rounds using Mini-Bead beater (Biospec, Bartlesville, OK, USA) at 4,800 rpm. The samples were centrifuged at 21,000 × g for 5 min. Supernatants were removed and stored at −80 °C until HPLC analysis. Samples were run similar to c-di-GMP (46 (link)) on an AB SCIEX TRIPLE QUAD 4500MD and peaks were quantified at 260 nm.