In vitro neuromuscular junction functionality_updated
Abolition of contractile activity in in vitro neuromuscular junctions within multi-nodal microfluidic chips using alpha-Bungarotoxin. Chips seeded as described above were allowed to mature for 21 days after seeding. Chips were selected for high contractile activity of myotubes that had visual contact with axons (more than 1 contraction per minute over 5 minutes with at least 1 contraction in each minute, the day prior to the experiment). Video of pre-experiment activity was recorded for 1 min followed by counting of the total number of contractions in 10 min. The cells were allowed to recover for 1 h at 37°C, 5% CO2 before addition of 1:100 of α-BTX at a final concentration of 1.25μM or of 0.75μl sterile PBS and incubation of 10 min at 37°C, 5% CO2. The α-BTX was only added to the central well containing the myotubes, which was fluidically isolated by hydrostatic pressure throughout the incubation. Another 1 min video of activity after intervention was recorded before counting the total number of contractions in 10 min. Cells were fixed immediately after the final count. For quantitative analysis the experiment was repeated with 10 min video being recorded as baseline followed by 1 h recovery at 37°C, 5% CO2 and treatment. Another 10 min video was recorded after treatment, and blinded quantification was carried out for both.