| Assay Method Information | |
| | Measurement of the GluN2B Potency (pH=6.9) |
| Description: | The GluN2B potency and pH dependence of NP10679, NP10309, and other compounds in Tables 1 and 2 were evaluated on human GluN1-la/GluN2B receptors (hereafter GluN1/GluN2B) expressed in Xenopus laevis oocytes by measuring the IC50 values at pH 6.9 and 7.6, respectively.Two Electrode Voltage-Clamp Recordings from Xenopus laevis OocytesStage V-VI Xenopus laevis unfertilized oocytes were purchased from Ecocyte (Austin, Texas) and injected with 5 ng of GluN1 and 10 ng of GluN2B cRNAs. The cDNAs for human GluN1 and GluN2B, encoding NCBI reference sequences NM_007327.3 and NM_000834.3, respectively, were linearized and cRNAs made as previously described (Traynelis et al., J Neurosci, 1998, 18(16):6163-75). After injection, the oocytes were incubated in Barth's culture solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 10 mM HEPES, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2), 10 U/mL PenStrep, and 0.1 mg/mL gentamycin, pH 7.4) at 18° C. Two electrode voltage-clamp (TEVC) recordings were made at 22-23° C., 2-7 days after the injection, using Warner OC725C amplifiers (VHOLD=−40 mV). Briefly, the oocytes were perfused in a recording solution (90 mM NaCl, 1 mM KCl, 10 mM HEPES, 0.01 mM EDTA, and 0.5 mM BaCl2) adjusted to either pH 6.9 by addition of NaOH or HCl, respectively (pH 6.9 solutions were prepared by addition of HCl to pH 7.6 solutions to maintain an equal concentration of Na+ ions in both solutions). |
| Affinity data for this assay | |
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