N palmitoyl d erythro sphingosine

Transepidermal water loss (TEWL) was measured using a VapoMeter (Delfin Technologies, Finland). Two sites per mouse were used for TEWL measurements. TEWL values were determined by triplicate measurements and the mean was defined as the TEWL value of every measurement site. After 3 days with skin washes as described above, TEWL was measured and mice were distributed into groups, so that the average TEWL of all groups was similar. On the fourth day, 2 × 10⁸ CFU (4 × 10⁷/cm²) of bacteria or 2.7 μg Sph protein or 50 μg ceramide d18:1/16:0 (N-palmitoyl-D-erythro-sphingosine, Avanti Polar Lipids, Inc.) were applied onto the compromised skin. TEWL was measured again 24 h later. For qRT-PCR of ceramide synthesis enzymes, skin tissues at the application sites were cut and saved in 1 ml of RNAlater solution (Invitrogen) at 4 °C. 50 mg tissue was cut up into small pieces, mixed with 1 ml of QIAzol Lysis Reagent and homogenized using Lysing matrix A (MP Biomedicals) twice at 1800 oscillations/min for 1 min in a FastPrep 96 homogenizer. RNA was extracted following the Qiazol Handbook (Qiagen). Then, the air dried RNA pellet was purified using an RNeasy Plus kit (Qiagen) according to the manufacturer’s protocol. qRT-PCR was performed with a SuperScript III Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen). See Table S2 for oligonucleotides.

Ceramides were extracted and analyzed via an liquid chromatography-electrospray ionization/multistage mass spectrometry system (API 3000 PE Sciex; Spectralab Scientific) in positive ionization as formerly described (Ramírez et al., 2013 (link)). Concentrations were measured by multiple reaction monitoring experiments using N-heptadecanoyl-D-erythrosphingosine (C17- ceramide) as an internal standard (50 ng/ml). The method was linear over the range from 2 to 600 ng/ml using as patrons N-palmitoyl-D-erythro-sphingosine (C16 ceramide) and N-stearoyl-D-erythro-sphingosine (C18 ceramide; Avanti Polar Lipids).