3h histamine

The membrane suspension, ³H-histamine (PerkinElmer, USA), and cold ligand (histamine) were diluted with the assay buffer (20 mM HEPES pH7.5, 150 mM NaCl). Briefly, 100 μL of the reaction mixture was dispensed in triplicate in a 96-well microplate. Membrane proteins (300 μg) were added to each well. The final concentrations of ³H-histamine were 200, 100, 50, 25, 12.5, 6.25, and 3.13 nM. To achieve non-specific binding, the experiment was performed in the presence of a cold ligand at a 1000-fold concentration of the hot ligand. The reaction mixture was incubated at room temperature (22–24 °C) for 1 h. The membrane was harvested on a glass fiber Filtermat B (PerkinElmer, USA) filter paper presoaked in 0.3% polyethylene imine using a FilterMate cell harvester (PerkinElmer, USA). The filter paper was washed with distilled water and dried. The solid scintillator, MeltiLex B/HS (PerkinElmer, USA), was melted on the filter paper. Radioactivity was detected using a Microbeta 2 system (PerkinElmer, USA). Specific binding was determined by subtracting nonspecific binding from total binding.

Fetal bovine serum (FBS, #16170078) was obtained from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Penicillin/streptomycin (P/S) was purchased from GE Healthcare (Uppsala, Sweden). Dulbecco’s Modified Eagles Medium (DMEM, #41966-029), Dulbecco’s phosphate-buffered saline (DPBS, #15326239), 0.05% Trypsin-EDTA (#11580626), and Hanks’ Balanced Salt Solution (HBSS, #11560456) were bought from Thermo Fisher Scientific (Waltham, MA, USA). Linear poly-ethylenimine (PEI, 25-kDa, # 23966-1) was obtained from Polysciences (Warrington, PA, USA). G418 (#108321-42-2) and isoprenaline (#I6504, (R)-3,4-Dihydroxy-α-(isopropylaminomethyl)benzyl alcohol hydrochloride) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Zeocin (#ZEL-43-05) was purchase from InvivoGen (San Diego, CA, USA). Black 96-well plates (#655086) were purchased from Greiner Bio-One (Frickenhausen, Germany). N^α-[methyl-³H]histamine (#NET1027250UC), [³H]histamine (#NET732250UC), Microscint-O scintillation liquid (#6013611), GF/C filter plates (#6055690) and MicrobetaWallac Trilux scintillation counter were purchased from PerkinElmer (Groningen, The Netherlands).