Ltb4 d4

HPLC-grade water, methanol, acetonitrile, and isopropyl alcohol were purchased from J.T. Baker (Avantor Performance Material, Inc., Center Valley, PA, USA). Acetic acid and ammonium acetate were obtained from Sigma-Aldrich (St. Louis, MO, USA). The eicosanoid standards were as follows: 14,15-EET-d11, 5(S)HETE-d8, LTB₄-d4, PGE₂-d4, PGD₂-d4, and AA-d8 (Cayman Chemical, Ann Arbor, MI, USA). A Strata-x 33-µm polymerized solid reverse-phase extraction column (cat # 8B-S100-UBJ) was purchased from Phenomenex (Torrance, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, and trypsin-ethylenediaminetetraAcetic acid (EDTA) were purchased from HyClone Laboratories Inc. (Logan, UT, USA). Escherichia coli LPS and Griess reagent were obtained from Sigma Chemical Co. (St Louis, MO, USA).

Cell-free culture media (0.5 ml) were mixed with 2 ng of deuterated internal standard solutions, centrifuged (12000× g, 3 min), mixed with 6 ml of 0.1% acetic acid, and loaded onto Oasis® PRiME hydrophilic lipophilic balance (HLB) solid-phase lipid extraction cartridge (60 mg, 3cc, (Waters, Milford, MA, USA) catalogue number 186008056). The cartridge was washed with 2 ml of 15% methanol containing 0.1% formic acid and the lipids were sequentially eluted with 500 μl of anhydrous methanol and 500 μl of acetonitrile. The resulting samples were mixed, concentrated by evaporation of the solvent under gentle stream of nitrogen, and stored at −80 °C. The target eicosanoids were identified by triple quadrupole UPLC-MS/MS (Shimadzu 8040, Kyoto, Japan) under previously reported run conditions [17 (link)] and quantified by comparing their MS, MS/MS, and UPLC (retention times, peak areas) data with the data obtained for deuterated internal standard compounds (6-keto PGF_1α-d4 (cat.no. 315210), TXB₂-d4 (cat.no. 319030), PGF_2α-d4 (cat.no. 316010), PGE₂-d4 (cat.no. 314010), PGD₂-d4 (cat.no. 312010), LTB₄-d4 (cat.no. 320110), 12(S)-HETE-d8 (cat.no. 334570) (Cayman Chemical, Ann Arbor, MI, USA) of the same classes using Lipid Mediator Version 2 software package (Shimadzu, Kyoto, Japan).

For lipid inflammatory mediators analysis, resting or IFNγ-primed BMDMs monolayers cultured in 6-well plates were infected with Mtb at a MOI of 2:1. At indicated time points, cell supernatants were collected, methanol was added at a final concentration of 30%, and samples were transferred at –80°C. Samples were thawed, supplemented with 2 ng per sample of LxA4-d5, LTB4-d4, and 5-HETE-d8 (Cayman Chemical), and centrifuged at 5000 rpm for 15 min at 4°C. Cleared supernatants were submitted to solid-phase extraction using Oasis HLB 96-well clusters, and lipid inflammatory mediators were analyzed by LC/MS/MS as previously described (Le Faouder et al., 2013 (link)) on an Agilent LC1290 Infinity ultra-high-performance liquid chromatography system coupled to an Agilent 6460 triple quadrupole mass spectrometer (Agilent Technologies) equipped with electrospray ionization operating in the negative mode. Reverse-phase ultra-high-performance liquid chromatography was performed using a ZORBAX SB-C18 column (inner diameter: 2.1 mm; length: 50 mm; particle size: 1.8 µm; Agilent Technologies) with a gradient elution, and quantifications were obtained using Mass Hunter software.