We designed a custom in-house optical stimulation apparatus consisting of a single 700 mA 470 nm LED enclosed in a 3D-printed dark box (Lulzbot 5.0) and activated using programmable parameters controlled by Multi Channel Experimenter software (Multi Channel Systems). Light density was determined using a PM121D optical power meter (Thorlabs) and characterized as a function of distance in order to determine an optimal firing rate-induction paradigm without causing noise interference. The LED was positioned 30 mm above the surface of the MEA, corresponding to a power density of 0.29 mW/mm2.
Multi channel experimenter software
Manufactured by MultiSciences Biotech
The Multi Channel Experimenter software is a digital data acquisition and analysis tool designed for conducting experiments with multiple channels. It provides a platform for capturing, visualizing, and processing data from various experimental setups.
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Lab products found in correlation
60pmea100 30ir ti, by MultiSciences Biotech (3 mentions)
Mea2100 system, by MultiSciences Biotech (2 mentions)
Pm121d, by Thorlabs (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
Laminin, by Merck Group (1 mentions)
Tc02 temperature controller, by MultiSciences Biotech (1 mentions)
Mea2100 lite system, by MultiSciences Biotech (1 mentions)
6 protocols using multi channel experimenter software
1
Custom Optical Stimulation Apparatus
2
Extracellular Recordings of Retinal Ganglion Cells
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The electrical activity of the RGCs was displayed and recorded with Multi Channel Experimenter software (Multi Channel Systems) at a sample rate of 25,000 Hz. Raw data was filtered with a second order Butterworth high pass filter with a cutoff at 200 Hz (filter 1) and a second order Butterworth low pass filter with a cutoff at 2,000 Hz (filter 2). A spike detector used the band pass filtered data from filter 1 and 2 to identify action potentials at a threshold of −20 μV. If necessary, noise channels had to be excluded from spike detection. Measurements of 60 s each were performed, recording either the spontaneous firing of the cells or their response to stimulation.
3
Extracellular Recording of Primary Neurons on MEAs
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MEAs (Multi Channel Systems, 60MEA200/10iR-Ti) consist of 59 titanium nitride (TiN) working electrodes with a diameter of 10 μm with 200 μm spacing between electrodes and one internal reference electrode. Prior to recording, MEAs were treated with oxygen plasma for 30 seconds using a PX-500 Plasma System (March Instruments), 100 μg/mL of poly-D-lysine overnight (Sigma, P0899), and 10 μg/mL Laminin (Sigma, L2020) for 2h prior to culture of primary neurons. For recording, culture medium was removed and 1 mL of warmed MEA recording solution85 (link),86 (link) was added to each MEA. Each culture was allowed to equilibrate with the MEA solution for 5 minutes in the 37°C incubator before recording. Recordings were conducted with a MEA2100-Lite-System (Multi Channel Systems) that was maintained at 37°C using the TC02 temperature controller (Multi Channel Systems). The Multi Channel Experimenter software (Multi Channel Systems) was used to record the extracellular potential at each electrode for 5 minutes using a sampling rate of 20 kHz. After recording, MEA recording solution was removed and the conditioned cell culture medium was returned to the MEA.
4
Acute Slice Recordings of Neural Activity
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Slices were placed above the electrodes, anchored, and positioned such that recording electrodes were in contact with the superficial layers of the SC near the midline of the brain. In all experiments, 6 × 10 perforated MEAs (100 µm spacing; 60pMEA100/30iR-Ti; Multi Channel Systems GmbH) were used. During the recording, gentle suction was applied to ensure proper contact with the recording electrodes and signal stability. Prior to the start of the recording, always 2 h after the cull, slices were allowed to settle for half an hour. Slices were continuously rinsed with fresh, carbogenated ACSF (2 ml min -1 ) heated to 32°C. All drugs were applied by bath perfusion. Signal was sampled at 20 kHz and acquired with the Multi Channel Experimenter software (Multi Channel Systems GmbH). 37
5
Electrophysiological Recordings of OPN
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Neuronal activity of the OPN was evaluated with the use of the two-well perforated MEA ex vivo technology (Belle et al., 2021) (link), according to a previously described procedure (Chrobok et al., 2021b (Chrobok et al., , 2021c) ) . Briefly, slices containing the OPN were transferred to the recording wells of the MEA2100-System (Multichannel Systems GmbH, Germany) and positioned upon the 6×10 recording array of the perforated MEA (60pMEA100/30iR-Ti, Multichannel Systems). Slices were constantly perfused with fresh recording ACSF (32 o C, 2 ml/min) and sucked down the array. Data were continuously collected with Multi Channel Experimenter software (sampling frequency = 20 kHz; Multichannel Systems).
6
Electrophysiological Responses to Orexin and GLP-1 in Brainstem Slices
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Experiments with the use of the MEA platform (Belle et al., 2021) were carried out at four daily time points on brainstem slices obtained from 23 rats (CD: n=11, HFD: n=12). Four to five slices were recorded at each time point in each diet. Slices were positioned in the recording wells of the MEA2100-System (Multichannel Systems GmbH, Germany) with the whole DMV localised upon the 6 × 10 recording array of a perforated MEA (60pMEA100/30iR-Ti, Multichannel Systems). Throughout the experiment, slices were perfused with fresh recording ACSF (2 ml/min), constantly bubbled with carbogen and heated to 32°C. Slices were allowed a minimum of 30 min to equilibrate before recordings were initiated. Data were acquired with MultiChannel Experimenter software (sampling frequency = 20 kHz; Multichannel Systems). Baseline recording was made for one hour. Then, drugs (orexin A, OXA 200 nM and GLP-1 1 µM) were diluted in 6 ml of fresh ACSF and bath applied in one hour intervals.
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