| Assay Method Information | |
| | S1P Transporter Assay |
| Description: | Transporter assays are vectorial and therefore require measurement of the transported analyte in different compartments. The S1P transporter SPNS2 only exports S1P, which obviates measuring uptake of S1P into transporter-expressing cells. Thus, transporter activity was determined by quantifying S1P release from whole cells expressing SPNS2. SPNS2 inhibitor potency was assessed using whole cell assays. HeLa cells expressing mouse SPNS2 were used to determine inhibitor potency (IC50). Cells were plated onto 12 well plates and assayed when the cell monolayers became confluent. Cell growth media (RPMI-1640 containing 10% fetal bovine serum) was replaced with 2 mL of serum-free media (RPMI-1640) containing fatty acid free bovine serum albumin (BSA) (0.2 % w/v) and supplemented with 4-deoxypyridoxine (DOP) (1 mM), NaF (2 mM), Na3VO4 (0.2 mM) to inhibit S1P degradation. Test articles (1 × 10-9 - 1 × 10-4 M) were assayed in duplicate or triplicate. After 18 hours, media was collected, an internal recovery standard (0.005 mL of 5 × 10-7 M deuterated (d7) S 1P in methanol) was added, the BSA was precipitated with trichloroacetic acid and the bound S1P extracted with methanol. S1P and S1P-d7 were measured by liquid chromatography mass spectrometry. |
| Affinity data for this assay | |
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