Sophion Bioscience A/S, Ballerup, Denmark.
Methods Mol Biol. 2021;2188:93-108. doi: 10.1007/978-1-0716-0818-0_5.
The automated patch clamp (APC) technology is used for increasing the data throughput of electrophysiological measurements, especially in safety pharmacology and drug discovery. Typically, electrical access to the cells are obtained using standard whole-cell formation by rupturing the membrane, thereby causing a rapid washout of cytosolic components. In contrast the perforated whole-cell configuration provides electrical access to the cell interior while limiting intracellular wash-out. This method allows for recordings of ion channels that are gated by intracellular modulators (e.g., ATP, cyclic nucleotides, or Ca), prevents channel current "run down," and maintains a physiological membrane potential for action potential recordings. Here we present some practical approaches to the use of perforated patch clamp for APC recordings. Our findings from these high-throughput, data-rich measurements (e.g., defining optimized concentrations and practical recommendations for four different perforating agents) can be more broadly applied to perforated patch clamp experiments in general (automated and manual), improving success rates, experimental conditions, and applications.
自动化膜片钳（APC）技术用于提高电生理测量的数据通量，特别是在安全药理学和药物发现中。通常，通过破坏细胞膜来获得细胞的电接触，从而导致细胞质成分的快速冲洗。相比之下，穿孔全细胞构型提供了对细胞内部的电接触，同时限制了细胞内的冲洗。这种方法允许记录由细胞内调节剂（例如 ATP、环核苷酸或 Ca）门控的离子通道，防止通道电流“衰减”，并维持动作电位记录的生理膜电位。在这里，我们介绍了一些在 APC 记录中使用穿孔膜片钳的实用方法。我们从这些高通量、数据丰富的测量中获得的发现（例如，定义四种不同的穿孔剂的优化浓度和实用建议）可以更广泛地应用于一般的穿孔膜片钳实验（自动和手动），提高成功率、实验条件和应用。
