FcR Blocking Reagent, Viobility™ 488/520 Fixable Dye, Ly-6G VioBlue® (REAfinity™, clone REA526), CD45 VioGreen® (REAfinity™, clone REA737), CD11c PE-Vio® 770 (clone REA754), and F4/80 APC (clone REA126) were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). Zymosan was obtained from InvivoGen (Toulouse, France). All other analytes and reagents were purchased from Sharlab (Sharlab S.L., Sentmenat, Spain), or Sigma-Aldrich (Sigma-Aldrich Chemical Co., Saint Quentin Fallavier, France). All the reagents were HPLC analytical grade, except those used for UHPLC-MSMS, which were of LC-MS grade. The 10 sphingolipids used as internal standards obtained from Sigma-Aldrich were manufactured by Avanti Polar Lipids under the trade name “Ceramide/Sphingoid Internal Standard Mixture I” and corresponded to C17 sphingosine (d17:1), C17 sphinganine (d17:0), C17 sphingosine-1-P (d17:1P), C17 sphinganine-1-P (d17:0P), lactosyl (ß) C12 ceramide (Lac18:1/12:0), C12 sphingomyelin (SM18:1/12:0), glucosyl (ß) C12 ceramide (Glu18:1/12:0), 12:0 ceramide (18:1/12:0), 12:0 ceramide-1-P (18:1/12:0P), and 25:0 ceramide (18:1/25:0) in ethanol solution. The 33 sphingolipids used as standards obtained from Sigma-Aldrich were solubilized in ethanol at a concentration of 25 µM and corresponded to deoxysphingosine (dSo = m18:1); deoxysphinganine (dSa = m18:0); sphingosine (So = d18:1); sphinganine (Sa = d18:0); sphingosine-1-P (d18:1P); sphinganine-1-P (d18:0P); glucosylsphingosine (GluSo); lysosphingomyelin (LysoSM); lactosylsphingosine (LacSo); N-acetylsphingosine (18:1/2:0); N-acetylsphinganine (18:0/2:0); ceramides: 18:1/14:0, 18:1/16:0, 18:1/18:0, 18:1/20:0, 18:1/22:0, 18:1/24:1, and 18:1/24:0; ceramide-1P: 18:1/16:0P; dihydroceramides: 18:0/16:0, and 18:0/24:0; glucosylceramides: Glu18:1/16:0 and Glu18:1/24:1; lactosylceramides: Lac18:1/16:0 and Lac18:1/24:1; sphingomyelins: SM18:1/14:0, SM18:1/16:0, SM18:1/18:0, SM18:1/18:1, SM18:1/20:0, SM18:1/22:0, SM18:1/24:1, and SM18:1/24:0.
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