Cell surface binding: HEK cells expressing human or rat P2X3, or human P2X2, and control cells were incubated with antibody supernatants for 1 h on ice, washed and incubated with rabbit anti-human Ig FITC (Sigma) for 1 h on ice, washed and analyzed by flow cytometry.
Inhibition of Ca++ signaling: Antibodies were tested for ability to inhibit α,β-meATP-induced calcium flux using a cell-based Ca2+ flux assay [41 (link)]. Briefly, canine Cf2Th cells were transfected with the appropriate expression vector in poly-lysine coated, black 384-well plates with clear bottoms (Costar). Cells were incubated for 22 h at 37°C then washed twice and loaded with a calcium indicator dye in HBSS containing 20 mM HEPES (Calcium 4 Assay kit, Molecular Devices), incubated for 1.5 h, then moved to a Flexstation II-384 (Molecular Devices) set at 32°C. After a 10-min temperature equilibration, the specific P2X3 agonist α,β-meATP (Sigma) was injected (at t = 20 s) and fluorescence was measured for 60 s (reading every 3 s). Data sets were analyzed using Prism 5.0 software (GraphPad Software, Inc).
Inhibition of Ca++ signaling: Antibodies were tested for ability to inhibit α,β-meATP-induced calcium flux using a cell-based Ca2+ flux assay [41 (link)]. Briefly, canine Cf2Th cells were transfected with the appropriate expression vector in poly-lysine coated, black 384-well plates with clear bottoms (Costar). Cells were incubated for 22 h at 37°C then washed twice and loaded with a calcium indicator dye in HBSS containing 20 mM HEPES (Calcium 4 Assay kit, Molecular Devices), incubated for 1.5 h, then moved to a Flexstation II-384 (Molecular Devices) set at 32°C. After a 10-min temperature equilibration, the specific P2X3 agonist α,β-meATP (Sigma) was injected (at t = 20 s) and fluorescence was measured for 60 s (reading every 3 s). Data sets were analyzed using Prism 5.0 software (GraphPad Software, Inc).
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