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. Author manuscript; available in PMC: 2016 Sep 21.
Published in final edited form as: J Immunol. 2015 Aug 7;195(6):2540–2551. doi: 10.4049/jimmunol.1501019

Figure 3. TLC purification of LC-MS fraction C identifies 6 active bands.

Figure 3

A. Listeria LC-MS fraction C was spotted at 150 μg/spot on a TLC plate and ran in the 40:25:3:6 system. The plate was then cut into strips and each individual strip was stained with one of four stains: Dittmer-Lester reagent (‘Phosphate’, to stain phosphate groups), α-naphthol (‘Sugar’, to stain sugar groups), MPA (‘General’, a general fatty acid tail stain), and ninhydrin (‘Amino’, to stain amino groups). These stains were used to identify 12 lipid bands for extraction B. The TLC plate-eluted lipid bands were incubated with 14S.6 cells and RAW (gray) or RAW-CD1d (black) cells in triplicate. Because of the silica plate extraction, weights of TLC bands reflected both silica and lipid weight. Therefore, the lipid bands were resuspended based on total weight and fold-dilutions of the stock lipids are displayed. IL-2 ELISAs were performed on culture supernatants after 16–18 h of co-culture. C and D. Collision MS/MS spectra for TLC Bands 1–3 were all identified as diphosphatidylglycerol (DPG) (C) and TLC band 4 was identified as phosphatidylglycerol (PG). E to H. Structures of the dominant DPG (E) and PG (F) lipids in the TLC bands, with comparison to mammalian (G) and Corynebacterium (H) PG demonstrating the different fatty acid tails found in these species. Panel B is representative of two independent experiments and presented as mean ± SEM.