D erythro sphingosine 1 phosphate

Methanol, formic acid and tert‐butyl methyl ether (MTBE) were purchased from Carlo Erba. Ammonium formate was purchased from Sigma‐Aldrich. d‐Glucosyl‐β1‐1′‐N‐tetracosanoyl‐d‐erythro‐sphingosine (GL1; d18:1/24:0) from Matreya LLC. d‐Glucosyl‐β1‐1′‐d‐erythro‐sphingosine (Lyso‐GL1; d18:1), d‐erythro‐sphingosine (Sph; d18:1), d‐erythro‐sphingosine 1‐phosphate (S1P; d18:1), and the internal standards (IS) C17‐d‐erythro‐sphingosine (C17B; d17:1), C17‐d‐erythro‐sphingosine‐1‐phosphate (C17‐S1P; d17:1) and N‐heptadecanoyl‐d‐erythro‐sphingosine (C17Cer; d18:1/17:0) were obtained from Avanti Polar Lipids.

The capillary sprouting assay was performed as previously described previously (Schulz et al., 2012 (link)). HDLECs were incubated in a 15 ml conical tube in presence of cytodex 3 beads (Sigma-Aldrich) at a ratio of 400 cells per bead in EGM-2MV medium (Lonza) for 4 h at 37°C with shaking every 20 min. Beads were then incubated in a cell flask at 37°C for 48 h in EGM-2MV medium. The HDLEC-coated beads were subsequently collected and labelled with 2 μM Cell Tracker Green dye (Invitrogen) for 30 min, embedded in 1 mg/ml Type I collagen hydrogel (Advanced Bio-Matrix) containing 2 μM D-erythro-sphingosine-1-phosphate (Avanti Polar Lipids), and cultured in black clear-bottom 96-well plates (Perkin-Elmer) in the presence or absence of 10,000 human iPS-derived macrophages-RFP per well. After 60 min of incubation, a 1:1 mix containing EGM2-Incomplete (EGM2 media without supplementation with EGF, FGF2 and VEGF-A; Lonza) and macrophage media (v/v) was added and the beads incubated for 2 days at 37°C. The beads were then fixed with 4% PFA for 30 min and washed twice with PBS. Each well was imaged with a Zeiss LSM 780 scanning confocal microscope and the number of sprouts per bead calculated.

Chemotaxis assays were performed using a transwell chamber (Costar) as previously described^{42 (link)}. The numbers of cells that migrated to the lower well after 2 h or 3 h incubation were counted using a MACSQuant flow cytometer (Miltenyi Biotec). The percent migration was calculated by the numbers of cells of a given subset that migrated into the bottom chamber divided by the total number of cells of that subset in the starting cell suspension, and multiplying the results by 100. D-erythro-sphingosine 1-phosphate was purchased from Avanti Polar Lipids. CCL19 and CXCL12 were purchased (R&D Systems). Fatty acid free bovine serum albumin (FAF-BSA) was purchased (Sigma-Aldrich).

pLNs (cervical, brachial, axillary and inguinal) were collected from wildtype C57BL/6, gently minced and digested with 1 mg/ml collagenase type I (Worthington Biochemical Corp., Lakewood, NJ, USA) and 2 μg/ml DNase I (Roche, Basel, Switzerland). The single-cell suspension was stained with anti-CD45 and anti-PNAd antibodies, and CD45^-/CD31⁺/PNAd⁺ high-endothelial cells were collected by FACS. 4 × 10⁴ high-endothelial cells were cultured in 100 μl serum-free EC-growth media (HuMedia-EG2; Kurabo, Osaka, Japan) with or without 10 μM FTY720 (non-specific S1PR antagonis) or W146 (S1PR1-specific antagonist) for 4 hr without S1P. After four hours, the cell culture supernatant was collected and high-endothelial cells were cultured further with media supplemented with charcoal-stripped FCS (homemade and checked by LC-MS/MS for depletion of S1P). One half of the cultures were cultivated without S1P, whereas the other half of the cultures was cultivated in the presence of 1 μM D-erythro-sphingosine-1-phosphate (Avanti Polar Lipids, Inc, Alabaster, AL, USA) for additional 20 hr. The concentration of CCL21 in the cell culture supernatants collected after four or 20 hr was quantified by ELISA using the mouse CCL21 DuoSet ELISA kit (R and D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol.