Development of Automated Voltage Clamp Assay for Kv1

Ca2+ is an important second messenger in cells of the immune system, including T lymphocytes (Feske 2007). Normally, its cytoplasmic concentration is maintained within a narrow range (e.g., <0.1 pM) by a variety of pumps that serve to sequester Ca2+ in intracellular stores such as the

Ky 1.3 IonWorks Automated Protocol

Add D-PBS (3.5 (iL) to each well of the PP and pump intracellular recording solution into its lower chamber

Determine the aperture resistance in each well of the PP

Add cell suspension (3.5 |iL) to each well of the PP and apply negative pressure to its lower chamber

| Wait 10 minutes

Determine the seal resistance in each well

Replace the solution in the lower chamber of the PP with intracellular recording solution containing amphotericin-B

| Wait 3 minutes

Replace the solution containing amphotericin-B with normal intracellular solution

Wait 10 minutes

Measure K+ current amplitude

Current Amplitude Ranges (nA)

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