Sphingosine 1 phosphate (s1p)

The murine antibody, 504B3, was purchased from Echelon Biosciences (Salt Lake City, UT, USA). All lipid species including, 1-oleoyl-LPA (18:1), 1-palmitoyl-LPA (16:0), 1-oleoyl-lysophosphatidylcholine (18:1 LPC), 1,2-dioleoyl-PA (18:1–18:1), and sphingosine 1-phosphate (d18:1) (Fig. 1) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Approximately 95–97% of LPA is lost when reconstituted without BSA [23 (link)] or suitable organic solvents. Therefore, stable lipids (i.e.,16:0 LPA) were dissolved in EtOH:H₂O (1:1 v/v), and unstable and highly hygroscopic lipids (i.e., 18:1 LPA, 18:1 LPC, 18:1–18:1 PA) were desiccated, then reconstituted in EtOH:H₂O (1:1 v/v solution) to prepare 5 mM stock solutions. 18:1 S1P was dissolved in methanol, desiccated, and reconstituted in 0.4% BSA according to the manufacturer’s instructions.
Fig. 1

Chemical structure of ligands used in the binding assays against the anti-LPA antibody, 504B3. Lipids varied by acyl chain length (16:0, 18:1 LPA), head group (18:1 S1P, 18:1 LPC), or the presence of a 2nd acyl chain (18:1–18:1 PA)

HPLC-grade methanol, acetonitrile, water, chloroform, and isopropyl alcohol were purchased from J.T. Baker (Phillipsburg, NJ, USA). HPLC-grade formic acid, hydrochloric acid, ammonium formate, and acetic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lipid standards used in this study were purchased from Larodan Fine Chemicals AB [Cholesteryl ester; CE (10:0), Monoacylglycerols; MG (15:1), Diacylglycerols; DG (8:0-8:0), Triacylglycerols; TG (11:1-11:1-11:1)] and Avanti Polar Lipids, Inc. [Phosphatidylcholine; PC (10:1-10:1), LysoPhosphatidylcholine; LPC (13:0), Phosphatidylethanolamine; PE (10:1-10:1), Lysophosphatidylethanolamine; LPE (14:0), Phosphatidylglycerol; PG (10:1-10:1), Lysophosphatidylglycerol; LPG (14:0), Phosphatidic acid; PA (10:1-10:1), LysoPhosphatidic acid; LPA (17:0), Phosphatidylserine; PS (10:1-10:1), Lysophosphatidylserine; LPS (17:1), Phosphatidylinositol; PI (16:0), Lysophosphatidylinositol; LPI (13:0), Ceramide; Cer (d18:1-12:0), Dihydroceramide; dCer (d18:0-12:0), Ceramide-1-phosphate; Cer1P (d18:1-12:0), Dihydroceramide-1-phosphate; dCer1P (d18:0-16:0), Sphingosine; SO (17:1), Sphinganine; SA (17:0), Sphingosine-1-phosphate; SO1P (17:1), Sphinganine-1-phosphate; SA1P (17:0), Sphingomyelin; SM (d18:1-12:0), Dihydrosphingomyelin; dSM (d18:1-12:0)].

Palmitic acid (PA) and oleic acid (OA) were dissolved in sterile DMSO then mixed with fatty acid-free bovine serum albumin (BSA) (all from Sigma) in serum-free medium for 15 min at room temperature^{2 (link),10 (link)}. The molecular ratio of BSA to PA was 1–5.3 when using 400 mM palmitic acid, and 1–2.65 when using 200 mM PA. Cells were then cultivated in this serum-free media containing PA for 6, 18 or 24 h prior to analysis. An equivalent percentage of DMSO was used as a vehicle control in the experiments. S1P (Avanti Lipids) was dissolved in serum-free medium containing fatty acid free BSA and sonicated in a water bath at 37 °C. S1P aliquots were prepared at 100 μM and stored at −80 °C.

S1P (Avanti Lipids) was dissolved in butanol:methanol (3:1) and sonicated in a water bath at 55 °C. S1P stock solution (1.32 mM) was further stored at −80 °C. Plates containing S1P were prepared by adding stock solution of S1P to cooled NGM after autoclaving⁷⁸ . For injections, 1 µM S1P was directly injected into the C. elegans gonad.

Reverse-osmosed ultrapure water was obtained from a Milli-Q Plus185 system (Millipore, Billerica, MA, USA). A MS grade methanol (MeOH), used for standards preparation, and acetonitrile (ACN) were obtained from Fisher Scientific (Hampton, NH, USA). Analytical grade ammonia solution (28%, GPR RECTAPUR^®) and acetic acid glacial (AnalaR^® NORMAPUR^®) were obtained from VWR Chemicals (Radnor, PA, USA).
Arachidonic acid, LPC 14:0, LPC 16:0, LPC 17:0, LPC 17:1, LPC 18:0, LPC 18:1, LPC 19:0, LPE 18:0, LPI 16:0, LPI 20:4, palmitoleic acid, oleamide, SPA-1P, sphinganine-C17, sphingosine, S1P, sphingosine d7, LPC 18:1 d7 were purchased from Avanti lipids (Birmingham, AL, USA). Bilirubin, lactic acid, lauric acid, oleic acid, adenosine, arginine, betaine, carnitine, cortisol, creatine, creatinine, hexanoylcarnitine, hippuric acid, hypoxanthine, leucine/isoleucine, phenylalanine, proline, propionylcarnitine, urea, carnitine d3, isoleucine d7, and palmitic acid d31 were purchased from Sigma-Aldrich (Darmstadt, Germany). phenylalanine d5 and valine d8 were purchased from Cambridge Isotope Laboratories Inc. (Andover, MA, USA).