Linomat 5 sample application unit

Recombinant lipases (1.4 ng/ml) or bacteria-conditioned media were diluted in 0.1 M sodium phosphate buffer (pH 6 unless stated otherwise) supplemented with equimolar concentrations (0.3 to 1.2 mM) of AFAs and cholesterol dissolved in DMSO or acetone, respectively. Upon overnight (18–22 h) incubation in glass vials at 37 °C with shaking, methanol (MeOH) and chloroform were added to stop the reaction and extract lipids according to the Bligh and Dyer protocol^{74 (link)}. The organic fraction was transferred to a fresh vial, dried, and resuspended in 2:1 (vol/vol) chloroform: MeOH. Lipid extracts were then applied to silica gel high-performance thin-layer chromatography (HPTLC) plates (silica gel 60 F₂₅₄, Merck) using a Linomat 5 sample application unit (CAMAG). Plates were developed in an automatic developing chamber ADC 2 (CAMAG) with a mobile phase system 90:10:1 (vol/vol/vol) petroleum ether: ethyl ether: acetic acid^{64 (link)}. Lipid spots were visualized in an iodine vapor chamber.

Phospholipids were isolated and quantified as described recently (20 (link), 21 (link)). Bacterial overnight cultures grown in MHB to an optical density at 600 nm (OD₆₀₀) of 0.05 were incubated in 100 ml fresh MHB until the exponential-growth phase (OD₆₀₀ of 0.5 to 1) was reached. After adjusting S. aureus strains to equal optical densities, the Bligh-Dyer method (37 (link)) was used to extract lipids with a chloroform-methanol-sodium acetate buffer (20 mM, pH 4.6) mixture (1:1:1 [vol/vol/vol]). Isolated lipids were vacuum dried, resuspended in chloroform-methanol (2:1 [vol/vol), and spotted onto silica gel 60 F254 high-performance thin-layer chromatography (HPTLC) plates (Merck, Darmstadt, Germany) with a Linomat 5 sample application unit (Camag, Berlin, Germany). Polar lipids were separated in an ADC 2 developing chamber (Camag, Berlin, Germany) with a chloroform-methanol-water (65:25:4 [vol/vol/vol]) running solvent. Phospholipids were detected by staining of phosphate groups with molybdenum blue, and the LysPG content was determined in relation to the total phospholipid content by densitometry analysis performed with ImageJ (http://rsbweb.nih.gov/ij/docs/guide/index.html) as described recently (20 (link), 21 (link)).