Envision 2100 plate reader

GPCR screening was performed according to the protocol reported by Chen et al.^{[36 (link)]} Briefly, HTLA cells (a HEK293-derived cell line containing a stably integrated tTa-dependent luciferase reporter and a β-arrestin2-TEV fusion gene) maintained in DMEM containing 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin were seeded in 96-well tissue culture plates (Eppendorf) and transfected with 200 ng of S1PR4 plasmid (Addgene plasmid #66499)^{[25 (link)]} for 20 h. The medium was then replaced with Dulbecco’s Modified Eagle Medium (DMEM) with 20 mM N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES) buffer and 1% Penicillin/Streptomycin, and independently treated with compounds 1 and 2, sphingosine-1-phosphate (S1P) (Fisher Scientific), and dimethyl sulfoxide (DMSO) solvent vehicle after 2 h. After 20 h of treatment, the luminescence was read by incubating each well for 20 min with 50 μl/well of Bright-Glo solution (Promega) diluted 20-fold in Dulbecco’s phosphate-buffered saline (DPBS) with 20 mM-HEPES. The luminescence was measured using Perkin Elmer EnVision 2100 plate reader.

The reporter cell line used to screen 2 for NF-κB activation (HEK-Blue^™ Null2-k; Invivogen, San Diego, California, USA) is a human embryonic kidney (HEK 293) cell line transfected with an NF-κB/AP-1-SEAP (secreted embryonic alkaline phosphatase) reporter gene. HEK-Blue^™ Null2-k was maintained according to the manufacturer’s instructions. Two-fold serial dilutions of the positive control, human TNF-α (100 ng/mL in DMSO; Cell Signaling Technology, Danvers, Massachusetts, USA) and of synthetic 2 (100 μg/mL in DMSO) were done in triplicate in a 96-well microplate. HEK-Blue^™ Null2-k cells were grown to approximately 80% confluence and the assay was carried out according to the manufacturer’s protocol with HEK-Blue^™ Detection (Invivogen, San Diego, California, USA), a cell culture medium that allows for colorimetric detection of SEAP. After 16 h of stimulation at 37°C 5% CO₂, SEAP activity was measured with Perkin Elmer EnVision 2100 Plate Reader at 630 nm.