Ds qi1 monochrome digital camera

Whole-cell patch clamp was used to record from iPSK3-derived spheroids cultured on glass covered slips. Cover slips were washed three times with extracellular recording solution containing (in mM) 136 NaCl, 4 KCl, 2 MgCl, 10 HEPES, and 1 EGTA (312 mOsm, pH 7.39) and were incubated in this solution at room temperature during recording. Glass electrodes (resistance 1–5 MΩ) were filled with intracellular solution containing 130 mM KCl, 10 mM HEPES, and 5 mM EGTA (292 mOsm, pH 7.20). Cells were visualized under phase contrast with a Nikon Eclipse Ti-U inverted microscope and attached DS-Qi1 monochrome digital camera. Recordings were made with an Axopatch 200B amplifier (Molecular Devices) and digitized with a Digidata 1440 A system (Molecular Devices). Ionic currents were recorded under a voltage clamp protocol (−60 mV to 135 mV in 15 mV steps, 250 ms in duration). Action potentials were recorded under a current clamp protocol (−100 pA to 200 pA in 20 pA steps, 800 ms in duration). Spontaneous post-synaptic currents were recorded under continuous voltage clamp at −80 mV for 2 min. Signals were filtered at 1 kHz and sampled at 10 kHz. Data was collected and analyzed using pCLAMP 10 software (Molecular Devices).

HeLa cells were loaded with Fura-2 AM (Invitrogen, Cat# F6774), dissolved in dimethyl sulfoxide and diluted to 2 μM in DMEM, by incubating at room temperature for 30 min. The cells were then washed three times for 5 min at each step. Fura-2 imaging was performed using a LAMBDA DG-4 lamp (Sutter Instrument Company) and a × 40/0.75 NA CFI Plan Fluor objective, with intermittent excitation using 340 and 380 nm filtered fluorescent light. The emitted light was collected with a Nikon DS-Qi1 monochrome digital camera after passing through a 510-nm emission filter.