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.采用α-银环蛇毒素（alpha-Bungarotoxin）处理多节点微流控芯片内的体外神经肌肉接头，以消除其收缩活动。按前述方法接种的芯片于接种后成熟培养21天。实验前一日，选取与轴突存在可视连接且肌管收缩活动活跃的芯片：要求5分钟内收缩频率不少于1次/分钟，且每分钟至少出现1次收缩。先录制1分钟的实验前活动视频，随后统计10分钟内的总收缩次数。将芯片置于37℃、5% CO₂环境中恢复1小时，随后分别加入终浓度为1.25μM的1:100稀释α-BTX，或0.75μl无菌磷酸盐缓冲液（PBS），并在37℃、5% CO₂条件下孵育10分钟。α-BTX仅添加至包含肌管的中央孔道，该孔道在整个孵育过程中通过静水压实现流体隔离。干预后再次录制1分钟的活动视频，随后统计10分钟内的总收缩次数。最终计数完成后立即对细胞进行固定。为开展定量分析，本实验重复设置如下流程：先录制10分钟视频作为基线数据，随后在37℃、5% CO₂环境中恢复1小时并进行处理；处理后再录制10分钟视频，随后对两组数据开展盲法定量分析。