Hyclone hepes buffered saline

High-throughput FLIPR assays were conducted using 384-well BioCoat (Corning) plates. Cells were washed briefly in assay buffer (HyClone™ HEPES-buffered saline [GE Life Sciences] comprising 149 mM NaCl, 4 mM KCl, 10 mM HEPES, and 5 mM glucose at pH 7.4 and 300 mOsm osmolality, supplemented with 2 mM CaCl₂ and 1 mM MgCl₂) prior to Calcium5 dye (Molecular Devices) loading for 1 h at RT. Following removal of excess dye, plates were placed in the FLIPR Tetra (Molecular Devices) chamber, and fluorescent Ca²⁺ signal was captured using ScreenWorks 4.0™ software (Molecular Devices).
To obtain neuronal α7 mediated FLIPR responses, we used 5 μM PNU-12059615—a selective drug that attenuates receptor desensitization. Tetrodotoxin, or TTX (500 nM) was included in the assay buffer to inhibit spontaneous action potentials.

High-throughput FLIPR assays were performed using 384-well BioCoat (Corning) plates. Assay buffer consisted of the HyClone™ HEPES-buffered saline (GE Life Sciences) described above, though prior to recordings, cells were loaded with Calcium 5 dye (Molecular Devices) for 1 h at room temperature. Plates were washed to remove excess dye, and then placed in the FLIPR Tetra (Molecular Devices) chamber. Recordings were captured using ScreenWorks 4.0™ software (Molecular Devices). Obtaining robust α7-mediated FLIPR responses required the presence of a selective PAM to attenuate desensitization, either 50 μM NS-1738 ^{70 (link)} for HEK293T cells or 5 μM PNU-120596 ^{15 (link)} for neurons. Neuronal FLIPR responses were also obtained in the presence of tetrodotoxin, or TTX (500 nM), to inhibit the firing of action potentials.