Microscint o

Binding reactions were set up in a 96 well flat bottom microtiter plate in a total volume of 250 μl per well. Reactions consisted of either 25 μg of membrane prep, [³H] ryanodine (concentrations 0.01–40 nM) and 2 μl of DMSO in a binding buffer containing 0.01% Pluronic (1.5M KCl, 10 mM ATP, 1.38 mM CaCl₂, 10 mM HEPES pH 7.4) or 25 μg of membrane prep, [³H] ryanodine (0.01–40 nM), 2 μl of ryanodine in DMSO (final concentration 10 μM) in 0.01% Pluronic. The reactions were incubated at room temperature for 2 h. The 96 samples were then loaded onto a 96 well filter plate pre-treated with 50 μl of 0.1% polyethylenimine, using a 96 well plate harvester (Brandel, MD, USA). Each filter with bound membranes was then washed 3 times with 250 μl of wash buffer (150 mM KCl, 10 mM HEPES pH 7.4 and left overnight at room temperature to dry. Each well on the plate was then filled with 50 μl MicroScint™-O (PerkinElmer, MA, USA) and the filter plate loaded into a TopCount NXT™ Micro-plate Scintillation counter (PerkinElmer, MA, USA). Specific binding and binding kinetics values (equilibrium dissociation constant K_d and Binding capacity/receptor density B_max) were calculated using GraphPad Prism v5.5 (GraphPad, CA, USA).

Sorted blood DC subsets and monocytes were adjusted to 2.5 × 10⁵ cells/ml in cRPMI and a three-fold dilution series of each population was prepared. Allogeneic PBMCs were added (5 × 10⁵ cells/well) at responder to stimulator cell ratios ranging from 2:1 to 162:1. Pokeweed mitogen (Sigma, Poole, UK) and cRPMI were added to wells containing only PBMC as positive and negative controls, respectively. After 72 h incubation at 37 °C in a humidified 5% CO₂ atmosphere, cells were pulsed with 1 μCi/well ³H-thymidine (GE Healthcare, Little Chalfont, UK) and incubated for a further 24 h. Cells were harvested onto filter mats using a Harvester 96 Mach III (TomTec Inc, Hamden, USA) and ³H-thymidine incorporation measured by addition of 25 μl/well Microscint O and counting on a MicroBeta2 (link) Plate Counter (both Perkin Elmer, High Wycombe, UK).

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).