USC SCAP-T Protocols

USC Cell Collection Protocol

Version 2

Electrophysiology and cell collection.

Healthy cortical pyramidal neurons (Layers II-V) or specified cell types from embryonic brain and spinal cord were visually identified with an infrared Dodt gradient contrast system. Patch pipettes (6-10 MOhm; 1.2 mm O.D.) were filled with intracellular solution (total of 5µl; Kgluconate 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 in the patch pipette was set with an automatic pressure control unit (ez-gSEAL 100b, Neobiosystem, USA). Before reaching the cell, positive pressure (50-70 mmHg) was applied, and once the pipette touched the cell membrane, gentle suction (-15 to -30 mmHg) was applied to form giga-seal. The seal was allowed to stabilize for 1 minute, and membrane rupture to achieve the whole-cell configuration was obtained by applying a brief pulse of suction (-90 to -150 mmHg for 400 ms). Standard wholecell patch clamp recording was conducted using the EPC-10 amplifier (HEKA). Initially, spontaneous membrane potential changes were monitored in currentclamp mode, after which a series of current injections of varying step amplitude were conducted to elicit action potentials. The amplifier was then changed to voltage-clamp mode, and a series of voltage steps were applied to record voltage-gated ion currents (to generate current-voltage curves). Cells that had leakage current larger than 50 pA or seal resistance less than 500 Mohm were excluded. In current-clamp mode, holding current was set to 0 pA or the value that would hold membrane potential at ~-60 to -70 mV. From the holding current (0.2 sec), square steps of current were injected for 2 seconds to elicit action potentials, followed by a return to the holding current (0.2 sec). All recordings have been corrected for liquid junction potential of 14.56 mV for K-gluconate pipet solution. A standard P/4 protocol was used to eliminate capacitive transients and leak currents in voltage-clamp mode. 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 applying positive pressure (100 mmHg). The tube was then flash frozen for storage in liquid nitrogen.