Gf b filter plate

Foetal calf serum (FCS) was obtained from PAA Laboratories (Wokingham, UK). Furimazine was purchased from Promega (Southampton, UK). Bicinchoninic acid protein assay kit and white 96-well microplates were obtained from Thermo Fisher Scientific (Waltham, MA, USA). GF/B filter plates and Microscint-O were from PerkinElmer (Groningen, The Netherlands). CA200645 was obtained from HelloBio (Bristol, UK). The synthesis of AV039 was described in Vernall et al. as compound 19 [34 (link)], while the synthesis of XAC-S-ser-S-tyr-X-BY630 (compound 27) and XAC-S-ser-S-tyr-X-BYFL (compound 28) was described in Vernall et al. 2013 [33 (link)]. PSB-11 and MRS1220 were purchased from Tocris Bioscience (Bristol, UK), and NECA was obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands). [³H]8-Ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]-purin-5-one ([³H]PSB-11) was kindly donated by Prof. C.E. Müller (University of Bonn, Germany) and its synthesis described in Müller et al. [35 (link)]. 1-Benzyl-8-methoxy-1H,3H-pyrido[2,1-f]purine-2,4-dione (compound 5) synthesis was described in Priego et al. as compound number 3 [36 (link)] and referred in Xia et al. [37 (link)] as compound number 5, while LUF7565 synthesis was described in Xia et al. as compound 27 [30 (link)]. All other chemicals and reagents were obtained from Sigma-Aldrich (Gillingham, UK).

Membranes were prepared from CHO-K1 cells expressing hS1P₁, hS1P₂, hS1P₃, hS1P₄, hS1P₅, rS1P₁, and rS1P₃ based on the methods of Mandala [10] (link) with modifications. Briefly, cells were washed with PBS, suspended in 1 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, and 1× Complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), and disrupted on ice using a dounce homogenizer. The homogenate was centrifuged for 10 min at 1000 g and the supernatant was centrifuged at 100,000 g for 60 min at 4°C. The pellet was suspended in 10 mM Tris-HCl (pH 7.4), 1 mM EDTA and stored at −80°C. ASP4058 and fingolimod-P were dissolved in dimethyl sulfoxide (DMSO) (Wako Pure Chemical Industries, Osaka, Japan) and then diluted with assay buffer (20 mM HEPES [pH 7.5], 100 mM NaCl, 10 mM MgCl₂, 0.1% fatty acid-free bovine serum albumin, and 5 µM GDP) to various concentrations. Membranes (20 µg) were mixed with test-compound solution (final concentration, 1% [v/v] DMSO) and 50 pM [³⁵S]-GTPγS (PerkinElmer, Waltham, MA, USA) in 150 µl of assay buffer. Membranes were incubated for 60 min at room temperature and collected onto GF/B filter plates (PerkinElmer), and then filter-bound radionuclides were measured using a TopCount NXT Microplate Scintillation and Luminescence Counter (PerkinElmer).

At ~80% confluence, cells were plated into 96-well plates (20,000 cells/well) and grown in the same medium and conditions as described above for 24 h. The cells were then serum starved for 4 h. After a 20 min incubation at 37 °C with 500 μM 3-Isobutyl-1-methylxanthine (IBMX), serum-free medium containing 500 μM IBMX and the appropriate agonists was added and then incubated for 10 min at 37 °C. The reaction was terminated by removing the medium and adding 60 μL of ice-cold assay buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA)). Plates were sealed with boiling mats and then boiled at 95 °C for 10 min. Plates were then centrifuged at 4000 rpm, 4 °C, for 10 min to remove debris. Fifty microliters of lysate was transferred to a 96-well plate. Lysate was incubated with ~1 pmol ³H-cAMP (PerkinElmer), and 7 μg protein kinase A (Sigma-Aldrich, St. Louis, MI, USA) with 0.05% bovine serum albumin (BSA). The assay was incubated at room temperature for 1 h. The reactions were then harvested onto GF/B filter plates (PerkinElmer) via rapid filtration by a 96-well plate Cell Harvester (Brandel) and washed 3 times with ice-cold water. Filter plates were dried, 40μL of Microscint-PS scintillation cocktail was added to each well, and then counted in a TopCount or Microbeta2 (PerkinElmer) microplate scintillation counter.

CHO membrane homogenates (10–20
μg protein) expressing hMOR, hKOR, or hDOR were incubated with
0.2–0.5 nM ³H-diprenorphine (PerkinElmer) and varying
concentrations of test ligands at 25^oC for 1 h, followed
by termination by rapid filtration through 96-well GF/B filter plates
(PerkinElmer). Plates were washed with ice-cold 50 mM Tris HCl pH
7.4 buffer, dried, and 40 μL of MicroScint-PS scintillation
cocktail (PerkinElmer) added. Bound radioactivity was measured using
a MicroBeta2450 scintillation counter (PerkinElmer). Assays were performed
on at least three separate occasions in duplicate. Data were analyzed
to provide Ki values as a measure of receptor affinity using GraphPad
Prism, v. 8.0.

Radioligand binding was performed as previously described [40 (link),41 (link)]. For the binding assay 50 µL of a dilution series of peptide was added to 50 µL of 3.3 nM [³H]DPDPE (K_d = 3.87 nM) or 2.35 nM of [³H]DAMGO (K_d = 1.07 nM) or 0.8 nM of [³H]U69,593 (K_d = 1.2 nM) in a clear 96 well plate. Next, 100 µL of membrane suspension containing 7 µg protein was added to the agonist wells and incubated for 90 min at room temperature. The reaction mixture was then filtered over a GF-B filter plate (Perkin Elmer) followed by four quick washes with ice-cold 50 mM Tris HCl. The plate was dried overnight, after which 50 µL scintillation fluid (Ultimagold uLLT) was added and radioactivity was counted on a Packard TopCount NXT scintillation counter. All working solutions were prepared in a radioligand assay buffer containing 50 mM Tris HCl, 10 mM MgCl₂, and 1 mM ethylenediaminetetraacetic acid at pH 7.4.