Cortical interneuron development is affected in 4H leukodystrophy 73 2 Supplementary Table 3. Primary antibodies for immunostaining Target Host Dilution Company Number CTIP2 GAD65/67 MAP2 Rat Rabbit Chicken 1:250 1:1000 1:5000 Abcam Sigma Millipore AB18465 G5163 AB5543 MBP Rat 1:500 Abcam AB7349 NF-200 Rabbit 1:1000 Sigma N4142 NF-H Mouse 1:100 Hybridomabank RT-97-s OLIG2 Rabbit 1:500 Millipore AB9610 SMI312 Mouse 1:1000 Eurogentech SMI-312P-050 Synaptophysin1 Guinea Pig 1:1000 Sysy 101-004 VGAT Rabbit 1:500 Sysy 131-002 Multi electrode array analysis Day 18 cortical neurons, generated as described above, were plated on PLO/Laminin coated MEA plates (Multi Channel Systems 24W300/30G-288) together with rat astrocytes (1:6 ratio) and cultured as described above. MultiChannel Headstage hardware (Multi Channel Systems) and Mulitwell-Screen software (Multi Channel Systems) were used to record baseline electrophysiological activity. Measurements were performed for 30 minutes (after 10 minutes incubation) while controlling the environment at 37°C and humidified 5% CO2. Raw data was sampled at 10 kHz and filtered using a high-pass 2nd order Butterworth filter with 100 Hz cut-off and low-pass 4th order Butterworth with 3500 Hz cut-off. The data was analyzed using Multiwell-Analyzer software (Multichannel Systems). Faulty electrodes were manually excluded from the analysis upon visual inspection. Spike detection threshold was set at -5.0 standard deviations from baseline. To start a burst at least 4 consecutive spikes had each to be less than 50 ms apart. Burst events needed a minimal duration of at least 50 ms. If the spikes within a burst are more than 50 ms apart a burst ends, new burst can only start 100 ms after the last burst. Network burst detection was set on 6 (=50%) or more participating channels of which at least 3 (25%) simultaneous. Results were exported and visualized using RStudio, SPSS was used for statistical testing. To measure the effect of different compounds on neuronal activity in 4H and control cells, cells were treated with either APV (50 µM), DNQX (10 µM), TTX (1 µM), GABA (10 µM) or Bicuculin/Gabazine (30 µM/20 µM), at 14 weeks post plating. In addition, to one well of each line the corresponding solvent (DMSO or H2O) was added in the same volume as the
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