Kolb I, Stoy W A, Rousseau E B, Moody O A, Jenkins A, Forest C R
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, 30332 USA.
Colleges of Nanoscale Science and Engineering, SUNY Polytechnic Institute, Albany, NY, 12203 USA.
Sci Rep. 2016 Oct 11;6:35001. doi: 10.1038/srep35001.
Patch-clamp recording has enabled single-cell electrical, morphological and genetic studies at unparalleled resolution. Yet it remains a laborious and low-throughput technique, making it largely impractical for large-scale measurements such as cell type and connectivity characterization of neurons in the brain. Specifically, the technique is critically limited by the ubiquitous practice of manually replacing patch-clamp pipettes after each recording. To circumvent this limitation, we developed a simple, fast, and automated method for cleaning glass pipette electrodes that enables their reuse within one minute. By immersing pipette tips into Alconox, a commercially-available detergent, followed by rinsing, we were able to reuse pipettes 10 times with no degradation in signal fidelity, in experimental preparations ranging from human embryonic kidney cells to neurons in culture, slices, and in vivo. Undetectable trace amounts of Alconox remaining in the pipette after cleaning did not affect ion channel pharmacology. We demonstrate the utility of pipette cleaning by developing the first robot to perform sequential patch-clamp recordings in cell culture and in vivo without a human operator.
膜片钳记录技术能够以前所未有的分辨率进行单细胞电学、形态学和遗传学研究。然而,它仍然是一项费力且低通量的技术,对于大规模测量(如大脑中神经元的细胞类型和连接特性表征)而言,在很大程度上不切实际。具体而言,该技术受到每次记录后手动更换膜片钳移液管这一普遍做法的严重限制。为了克服这一限制,我们开发了一种简单、快速且自动化的玻璃移液管电极清洁方法,使其能够在一分钟内重复使用。通过将移液管尖端浸入市售洗涤剂爱尔康诺克斯(Alconox)中,然后冲洗,我们能够在从人胚胎肾细胞到培养中的神经元、脑片和体内等各种实验制剂中,将移液管重复使用10次,且信号保真度无下降。清洁后移液管中残留的痕量爱尔康诺克斯无法检测到,这并不影响离子通道药理学。我们通过开发第一台无需人工操作即可在细胞培养和体内进行连续膜片钳记录的机器人,证明了移液管清洁的实用性。