Net974001mc

Adenosine A_2B receptor competition binding experiments were carried out in a polypropylene 96-well plate. In each well was incubated 20 μg of membranes from Euroscreen HEK-A_2B cell line and prepared in Plataforma de Screening de Farmacos (USEF) (Lot: A007/22-02-2016, protein concentration = 3211 μg/mL), 25 nM [³H]-DPCPX (164 Ci/mmol, 1 mCi/mL, PerkinElmer NET974001MC) and compounds studied and standard. Non-specific binding was determined in the presence of NECA 1000 μM (Sigma E2397). The reaction mixture (Vt: 250 μl/well) was incubated at 25 °C for 30 min, 200 μL was transferred to GF/C 96-well plate (Millipore, Madrid, Spain) pre-treated with binding buffer (Tris-HCl 50 mM, EDTA 1 mM, MgCl₂ mM, Bacitracin 100 μg/μl, adenosine deaminase 2 U/mL, pH = 6.5), after was filtered and washed four times with 250 μl wash buffer (Tris-HCl 50 mM, EDTA 1 mM, MgCl₂ 5 mM, pH = 6.5), before measuring in a microplate beta scintillation counter (Microbeta Trilux, PerkinElmer, Madrid, Spain).

Adenosine A₁ receptor competition binding experiments were carried out in a multiscreen GF/C 96-well plate (Millipore, Madrid, Spain) pre-treated with binding buffer (Hepes 20 mM, NaCl 100 mM, MgCl₂ 10 mM, 2 U/ml adenosine deaminase, pH = 7.4). In each well was incubated 5 μg of membranes from Euroscreen CHO-A₁ cell line and prepared in Plataforma de Screening de Farmacos (USEF) (Lot: A002/13-04-2011, protein concentration = 5864 μg/mL), 1 nM [³H]-DPCPX (120 Ci/mmol, 1 mCi/mL, PerkinElmer NET974001MC) and compounds studied and standard. Non-specific binding was determined in the presence of R-PIA 10 μM (Sigma P4532). The reaction mixture (Vt: 200 μl/well) was incubated at 25 °C for 60 min, after was filtered and washed four times with 250 μl wash buffer (Hepes 20 mM, NaCl 100 mM, MgCl₂ 10 mM pH = 7.4), before measuring in a microplate beta scintillation counter (Microbeta Trilux, PerkinElmer, Madrid, Spain).