USC SCAP-T Protocols

USC Cell Collection Protocol Version 1

Electrophysiology and cell collection. Healthy cortical pyramidal neurons and interneurons (Layers II-V) were visually identified with infrared Dodt gradient contrast system. Patch pipettes (6-10 MOhm; 1.2 mm O.D.) were filled with intracellular solution (Total of 5µl; K-gluconate 130 mM; KCl 2 mM; CaCl2 1 mM; MgATP 4 mM; GTP 0.3 mM; phosphocreatine 8 mM; HEPES 10 mM; EGTA 11 mM; pH 7.25 and 300 mOsm) containing RNase inhibitor (0.4 U/µl; RNase inhibitor, Clontech) and Lucifer Yellow (0.625 µg/ml; Invitrogen, USA). Pressure control of the patch pipette was done with an automatic pressure control unit (ezgSEAL 100b, Neobiosystem, USA). Before reaching the cell, a positive pressure (50-70 mmHg) was applied, and once the pipette touched the cell membrane, a gentle suction (-15 to -30 mmHg) was applied to form giga-seal. The seal was allowed to stabilize for 1 minute and the membrane rupture for whole-cell was done through a brief suction (-90 to -150 mmHg for 400 ms). Standard whole-cell patch clamp (EPC-10 amplifier, HEKA) was performed, and cells were patch clamped in current-clamp mode for measuring spontaneous membrane potential change, followed by a series of current injections for triggered action potentials and in voltage-clamp mode for measuring currents from voltage-gated ion channels. Following recording, the cytoplasmic content from the soma was collected into the pipette with a strong negative pressure (-200 to -250 mmHg). The content was expelled into a PCR tube by breaking the pipette tip and a positive pressure (100 mmHg). 

 

 

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