Whole-lysosome patch clamp recordings were done as previously described51 (link). Briefly, transfected cells were re-plated onto 12-mm coverslips ~36 hr after transfection. Lysosomes were enlarged with vacuolin-1 (1 μM, overnight) and were released from cells using glass pipettes. Polished glass pipettes used for patch-clamp recordings had resistance of 5–8 MΩs. Pipette solution (lumen) contained (in mM) 145 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, 10 MES and 10 glucose (pH adjusted to 4.6 with NaOH). Bath solution contained (in mM) 140 K-gluconate, 4 NaCl, 2 MgCl2, 0.39 CaCl2, 1 EGTA and 10 HEPES (pH adjusted to 7.2 with KOH). Recordings were done 48 - 72 hr after transfection. Signals were amplified and filtered at 1 KHz using a MultiClamp 700B amplifier, and digitized at 5 KHz with a Digidata 1400A digitizer, both controlled with Clampex 10.4 (from Molecular Devices). Lysosomes were held at 0 mV. Current-voltage relationships were obtained using a ramp protocol from −100 mV to +50 mV in 1 s. Data were analyzed using Clampfit 10.4 (Molecular Devices), Excel (Microsoft) and Origin (Origin Lab).
Digidata 1400a digitizer
Manufactured by Molecular Devices
Sourced in United States
The Digidata 1400A is a high-performance digitizer that converts analog signals into digital data for acquisition and analysis. It provides reliable and accurate data capture for a variety of applications.
Automatically generated - may contain errors
3 protocols using digidata 1400a digitizer
1
Whole-Lysosome Patch Clamp Recordings
2
Extracellular Recordings of Hippocampal Slices
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Extracellular field recordings were performed in the CA3 pyramidal layer of hippocampal slices of P16 mice with glass pipettes (Harvard Apparatus, MA, USA) containing ACSF. Spike frequency was recorded for 10 min (control period), then isoguvacine (10 μM; Sigma-Aldrich, MO, USA) was applied in the bath solution for 90 s (isoguvacine period). Following isoguvacine application, spike frequency was recorded for an extra 15 min (wash-out period). Signals were recorded with a low-noise multichannel DAM-80A amplifier (WPI, UK; low-pass filter 1 Hz; high-pass filter 3 kHz; gain ×1000) and digitized online with a Digidata 1400A digitizer (Molecular Devices, CA, USA). Recordings were analyzed using Clampfit 10.4 software (Molecular Devices, CA, USA). The spike detection threshold was defined as three times the standard deviation of the noise recorded in the bath solution. Spike frequency was calculated for control, isoguvacine, and wash-out periods. Slices for which wash-out spike frequency did not come back to control levels (±20% of control) were excluded from this study.
3
Whole-Lysosome Patch Clamp Recordings
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Whole-lysosome patch clamp recordings were done as previously described51 (link). Briefly, transfected cells were re-plated onto 12-mm coverslips ~36 hr after transfection. Lysosomes were enlarged with vacuolin-1 (1 μM, overnight) and were released from cells using glass pipettes. Polished glass pipettes used for patch-clamp recordings had resistance of 5–8 MΩs. Pipette solution (lumen) contained (in mM) 145 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 HEPES, 10 MES and 10 glucose (pH adjusted to 4.6 with NaOH). Bath solution contained (in mM) 140 K-gluconate, 4 NaCl, 2 MgCl2, 0.39 CaCl2, 1 EGTA and 10 HEPES (pH adjusted to 7.2 with KOH). Recordings were done 48 - 72 hr after transfection. Signals were amplified and filtered at 1 KHz using a MultiClamp 700B amplifier, and digitized at 5 KHz with a Digidata 1400A digitizer, both controlled with Clampex 10.4 (from Molecular Devices). Lysosomes were held at 0 mV. Current-voltage relationships were obtained using a ramp protocol from −100 mV to +50 mV in 1 s. Data were analyzed using Clampfit 10.4 (Molecular Devices), Excel (Microsoft) and Origin (Origin Lab).
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