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. 2014 Aug 19;111(35):12746–12751. doi: 10.1073/pnas.1404372111

Fig. 2.

Fig. 2.

Chirality of lipoxins, requirement for priming, and separate effects of TLR4 and P2X7 stimulation. (A) MS monitoring was set to multiple-reaction monitoring (MRM) transition 319(−) to 219(−) m/z for 15-HETE in the first period (Left column) and 351(−) to 115(−) m/z for lipoxins in the second period (Right column); intensity is expressed in counts per second, cps). Chiral separation of (Top row) 15(R)-HETE:15(S)-HETE standards at a 1:3 concentration and 15-epi–LXA4:LXA4 standards at a 1:3 concentration; (Middle row) RAW cell medium after 7.5 h KLA and final 30 min ATP; (Bottom row) RAW cell medium after 7.5 h KLA in the presence of 1 mM ASA and final 30 min ATP. Arrowheads indicate a species ∼12 s to the Right of LXA4. A total of ∼25 million cells in a T-75 tissue culture flask containing 5 mL medium was used for both conditions [this cell quantity is considerably higher than in other experiments due to decreased signal yielded in atmospheric pressure chemical ionization (APCI) mode]; n = 1. (B) RAW medium after (Upper Left) only KLA stimulation for 8 h; (Upper Right) no stimulation for 7.5 h before ATP for final 30 min; (Lower Left) 7.5 h KLA priming before ATP stimulation for final 30 min; (Lower Right) 7.5 h KLA priming in the presence of 1 mM ASA before ATP stimulation for final 30 min. Chromatogram traces represent 70-s scheduled monitoring of MRM transition 351(−) m/z to 115(−) m/z during nonchiral reverse-phase chromatographic separation on a scale of 100 arbitrary units (Insets are on a one order of magnitude lower scale); data represent one replicate of n = 3. (C, Left) Chromatograms (as in B) of RAW medium after 30 min KLA or ATP stimulation in the presence of 1 μM 15(R)-HETE; coelution with LXA4 and 15-epi–LXA4 commercial standards are indicated with a green asterisk; (Right) 15-HETE levels from saponified phospholipids of RAW cells after KLA stimulation for the indicated times over a 24-h period represent mean values of three separate experiments ± SEM; 15-HETE levels were increased with KLA stimulation vs. control (*P < 0.001).