Silica 60 f254 hptlc plates

Briefly, late stationary phase cells were used to extract the PLs from SF100 and its DAP-R variants, as described previously [15 (link),17 (link),18 (link)]. The major PLs (i.e., CL, PG, and PA, as well as a prominent glycolipid [GL] seen in all VGS) were separated by two-dimensional thin-layer chromatography (2D-TLC) (Silica 60 F254 HPTLC plates [Merck]). Identification of all the PL spots on the TLC plates was confirmed by comparison to known standards following exposure to iodine vapors and spraying with CuSO₄ (100 mg/mL) containing 8% phosphoric acid (v/v) and heated at 180 °C [15 (link),17 (link),18 (link)]. The quantification of individual PLs removed directly from TLC plates was performed by digesting with 70% perchloric acid (0.3 mL) at 180 °C for 3 h. The individual PLs were quantified by measuring the optical density at 660 nm using spectrophotometry as described before [15 (link),17 (link),18 (link)]. The PL results are expressed as their individual mean proportions (±SD), employing at least three independent experiments performed on different days.

To investigate potential correlates between mprF polymorphisms and CM features, PLs were extracted from study strains under specific test conditions as described [26] (link). The major CM PLs of S. aureus (PG; L-PG and cardiolipin [CL]) were separated by two-dimensional thin-layer chromatography (2-D TLC) using Silica 60 F254 HPTLC plates (Merck). Fluorescamine labeling (a fluorophore which binds only to positively charged PLs, such as L-PG, and which does not penetrate the outer CM leaflet), combined with ninhydrin staining localization, was used within the 2-D TLC plate assay to assess the translocation of L-PG between the inner-to-outer CM bilayer [13] (link), [17] (link), [26] (link). First-dimension chloroform-methanol–25% ammonium hydroxide (65∶25∶6, by volume) in the vertical orientation and second-dimension chloroform:water:methanol:glacial acetic acid:acetone (45∶4∶8∶9∶16, by volume) in the horizontal orientation were used for the separation of the PLs for further quantitation by phosphate estimation. PL identity was confirmed using known PL standards. For quantitative analysis, resulting isolated PLs (identified by iodine staining) were digested at 180°C for 3 h with 0.3 ml 70% perchloric acid and the oxidized derivatives quantified spectrophotometrically at OD₆₆₀.

Lipids were extracted from TSB-grown (10 ml) and serum-adapted (10 ml) cultures using a method based on that of Bligh and Dyer^{59 (link),60 (link)}. Cells were washed twice with PBS, twice with 0.1% Triton X-100 and twice with PBS to remove unincorporated serum-derived lipids^{35 (link)} before resuspension in 300 µl 2% NaCl. After addition of 0.2 mg ml⁻¹ lysostaphin, cells were incubated at 37 °C for 10 min. 1 ml chloroform-methanol (2:1; v/v) was added, vortexed for 2 min and incubated at room temperature for 30 min. Chloroform (300 µl) and 2% NaCl (300 µl) were added, and after centrifugation, the lower layer was recovered and evaporated at room temperature. Samples were resuspended in 100 µl chloroform-methanol (2:1; v/v) and separated by one-dimensional TLC. Equal volumes of lipid extracts were spotted onto silica 60 F254 HPTLC plates (Merck) and migrated with chloroform:methanol:ammonium hydroxide (30%) (65:30:4; v/v/v). After drying, TLC plates were sprayed with CuSO₄ (100 mg ml⁻¹) in 8% phosphoric acid and heated at 180 °C. Signal intensities were quantified using ImageJ.