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Using this device, purified plasmid DNA encoding hNEP was injected into hindlimb skeletal muscle of 6-week-old KunMing mice and electrostimulation (50 V/cm, 6 pulses, 20 ms per pulse) was applied to the syringe needles.
To ensure proper electroporation of the injected DNA constructs (1 2 µl) into the progenitor cells, five electrical square pulses of 45 V with a duration of 50 ms per pulse and 150 ms inter-pulse interval were delivered using tweezer-type electrodes connected to a pulse generator (ECM 830, BTX Harvard Appartus).
Fertilized eggs in 100 µl culture solution were mixed with 200 µl of 0.96 M mannitol and 50 µl of 0.25 0.5 µg/µl plasmid DNA solution, transferred to a cuvette with a 4 mm electrode gap and electroporated using the pulse generator ECM 830 (BTX, CA), according to a square pulse protocol (50 V and 16 ms per pulse).
The parameters were 10 pulses (25 ms per pulse, separated by 2 minutes, with a current of 100 V/cm).
320 μl dechorionated fertilized eggs were added and electroporated with a Gene Pulser Xcell System (BIO-RAD), using a square pulse protocol (50 V and 15 ms per pulse).
The different cortical areas were identified by a clinical examination of unit receptive fields and by noting the motor responses to intracortical microstimulation (13 18 biphasic pulses, 300 Hz, 0.2 ms per pulse, currents up to 50 µA).
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Prejunctional cells were stimulated with two kinds of current injections, the step currents (300 ms to 1 s duration) and a train of eight stimulating pulses at 20 to 70 Hz (3 ms duration per pulse).
We generated a series of stimuli that had identical numbers of 3.5 ms pulses per time, with pulse patterns that varied from single pulse to double pulse patterns by moving every other pulse closer to the preceding pulse.
The software generated biphasic electrical pulses (0.25 ms per phase) that were delivered at current amplitudes between 0.3 and 0.8 mA to stimulate the sciatic nerve.
Parameter 1 1 ms pulse width/49 ms intervals, Parameter 2 5 ms pulse width/45 ms intervals, Parameter 3 10 ms pulse width/40 ms intervals, Parameter 4 25 ms pulse width/25 ms intervals, Parameter 5 40 ms pulse width/10 ms intervals, Parameter 6 45 ms pulse width/5 ms intervals, Parameter 7 49 ms pulse width/1 ms intervals.
Experiments were acquired under continuous excitation (561 nm laser, 5 kW/cm on the sample) and pulsed activation (405 nm laser, 1 pulse of 10 ms per second, 0.01 kW/cm during the pulse on the sample).
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