Search and Browse
Download
Enter Data
|
Assay Method Information |
|
| | Electrophysiological Assay |
| Description: | Current record was obtained by an automated patch clamp system IonWorks Quattro (Molecular Devices Corporation) in Population Patch Clamp mode. The operation was conducted in accordance with the operating procedure of the system. A Dulbecco's phosphate buffer containing calcium and magnesium (Sigma) was used as an extracellular fluid, and a low Cl-buffer (100 mM K-gluconate, 40 mM KCl, 3.2 mM MgCl2, 5 mM EGTA, 5 mM Hepes, pH 7.3) was used as an intracellular fluid. A test compound was dissolved in dimethylsulfoxide (DMSO) to prepare a 30 mM stock solution, so as to produce 4-fold serial dilutions with the extracellular fluid for attaining a DMSO concentration of 0.3% in measurement.The hNav 1.7/β1/β2 cells cultured to a 70-80% confluent state in a T150 flask (Sumilon) were washed with PBS and subsequently with versene (Invitrogen Corp.), and collected by allowing to react with 0.05% trypsin (Invitrogen Corp.) at 37° C. for 3 minutes. After washing with a culture medium, the resultant cells were suspended in an extracellular fluid at a concentration of 2x10-6 cells/ml so as to be used for the measurement. The cell membrane was perforated by using an intracellular fluid including 100 ug/ml amphotericin B (Sigma). Current response was obtained at a sampling frequency of 10 kHz. Leakage current correction was performed by applying a step pulse of -110 mV before a test pulse. The membrane potential was fixed at -100 mV for 5 seconds immediately before applying the test pulse. In order to check the state-dependency of the inhibiting activity of a test compound, the test pulse was applied as follows: After applying a depolarization pulse of -10 mV for 5 msec., the potential was fixed at -100 mV for 200 msec., a potential (V1/2) at which approximately 50% of channels are inactivated was held for 2 seconds, and a depolarization pulse of -10 mV was applied for 50 msec. Such a test pulse was applied before adding the test compound and after cultivation of 5 minutes and 30 seconds with a solution of the test compound gradually added by 3.5 ul at each time. Since IonWorks Quattro has a measuring electrode head (E-head) and an agent supplying head (F-head) separated from each other, the membrane potential was not clamped during the addition and the cultivation of the test compound. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |
|
Home |
| |
Search |
| |
Deposit |
| |
SiteMap |
| |
About us |
| |
Email us |
| |
Info |
|
©2000 BindingDB. All rights reserved. |
|||||||||||||