Sutter p 1000 puller

Whole-cell recordings in current-clamp mode were performed in a temperature-controlled recording chamber (36–37°C) mounted on an inverted Eclipse-Ti microscope (Nikon) and using a MultiClamp 700B amplifier (Molecular devices, LLC). Current-command protocols and data acquisition were performed using pClamp 10.0 software and the Digidata 1550 interface (Molecular Devices, LLC). Data were lowpass-filtered at 3 kHz and sampled at 10 kHz. Patch electrodes, fabricated from thick borosilicate glass capillaries, have been made using a Sutter P-1000 puller (Sutter Instruments) to a final resistance of 4–6 MΩ when filled with the intracellular solution containing (in mM): 120 KGluconate, 25 KCl, 10 EGTA, 10 HEPES, 1 CaCl₂, 4 Mg-ATP, 2 Na-GTP, and 4 Na₂-Phosphocreatine (pH 7.4, adjusted with KOH). Cells were bath-perfused with a Krebs solution composed of (in mM): 129 NaCl, 5 KCl, 2 CaCl₂, 1 MgCl₂, 30 D-glucose, 25 HEPES, pH 7.3 with NaOH. Spontaneous action potentials were recorded in a gap-free mode while the presence of the Ih current was evaluated by applying long hyperpolarizing steps at different current intensities (−30 pA increments). Series resistance value was monitored during the experiment, and recordings with changes over 20% of their starting value were discarded.

Single-cell manipulation was performed using a pair of nanocapillary tips and microcapillary holders. The nanocapillaries were pulled from borosilicate glass capillaries (i.d. = 0.58 mm, o.d. = 1 mm, with filament) with a Sutter P-2000 puller (Sutter Instrument, Novato, USA) to make the nanospray tips suitable for MS ionization. A two-step pulling program was set as follows: HEAT = 350, FIL = 3, VEL = 30, DEL = 220, PULL = NA; HEAT = 350, FIL = 3, VEL = 40, DEL = 180, PULL = 120. The prepared nanocapillaries were characterized by SEM (JSM 7800F, JEOL Ltd., Tokyo, Japan). The microcapillaries were pulled from borosilicate glass capillaries (i.d. = 0.58 mm, o.d. = 1 mm, with filament) with a Sutter P-1000 puller (Sutter Instrument, Novato, USA) to make the microcapillary holders. The single-step pulling program was as follows: HEAT = 520, PUL = 100, VEL = 30, TIME = 250, PRESSURE = 500. The pulled capillaries were further processed with a Microforge (MF2, Narishige, Tokyo, Japan) to make the holders ∼1 μm open.