| Assay Method Information | |
| | Biological Assay |
| Description: | The QPatch HT system (Sophion Bioscience A/S, Denmark) was used with the conventional whole-cell configuration. This system is an automated, chip-based planar patch clamp device allowing for up to 48 parallel independent experiments in one experimental assay run. Cells are added to each well and drawn by suction onto a small aperture to obtain a Gigaohm seal between the cell membrane and treated silicon surface, and whole-cell recordings initiated after access is achieved by suction and/or voltage pulses. The QPatch HT uses static perfusion, whereby a small volume of recording solution or drug is added to a reservoir on the chip and the solution perfuses across the cell through quartz-lined microfluidic channels; this solution is removed by capillary action when the next sample application is made.CHO-Kv1.3 cells were prepared for experiments by dissociation from T175 cell culture flasks using trypsin-EDTA (0.05%), cells were kept in serum free media in the cell hotel on board the QPatch HT. These cells were sampled, washed and re-suspended in extracellular recording solution by the QPatch HT immediately before application to the recording well site on the chip. Experiments were performed using the following solutions; extracellular solution contained (in mM); 150 NaCl, 10 KCl, 1 MgCl2, 3 CaCl2), 10 Glucose and 10 HEPES (pH 7.4, NaOH) and intracellular solution contained (in mM); 20 KCl, 120 KF, 10 HEPES, 10 EGTA, 5 ATP (pH 7.2, KOH).The potency (Inhibitory Concentration 50%, IC50) of synthesized compounds against Kv1.3 was determined from concentration-response relationships established by cumulatively applying four escalating concentrations of test compound to an individual cell and a minimum of N≥3 individual cells of data per compound were used to generate the IC50 value. |
| Affinity data for this assay | |
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