Rapedius Markus, Obergrussberger Alison, Humphries Edward S A, Scholz Stephanie, Rinke-Weiss Ilka, Goetze Tom A, Brinkwirth Nina, Rotordam Maria Giustina, Strassmaier Tim, Randolph Aaron, Friis Søren, Liutkute Aiste, Seibertz Fitzwilliam, Voigt Niels, Fertig Niels
Nanion Technologies GmbH, Munich, Germany.
Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Cambridge, United Kingdom.
Front Mol Neurosci. 2022 Aug 22;15:982316. doi: 10.3389/fnmol.2022.982316. eCollection 2022.
Fluoride has been used in the internal recording solution for manual and automated patch clamp experiments for decades because it helps to improve the seal resistance and promotes longer lasting recordings. In manual patch clamp, fluoride has been used to record voltage-gated Na (Na) channels where seal resistance and access resistance are critical for good voltage control. In automated patch clamp, suction is applied from underneath the patch clamp chip to attract a cell to the hole and obtain a good seal. Since the patch clamp aperture cannot be moved to improve the seal like the patch clamp pipette in manual patch clamp, automated patch clamp manufacturers use internal fluoride to improve the success rate for obtaining GΩ seals. However, internal fluoride can affect voltage-dependence of activation and inactivation, as well as affecting internal second messenger systems and therefore, it is desirable to have the option to perform experiments using physiological, fluoride-free internal solution. We have developed an approach for high throughput fluoride-free recordings on a 384-well based automated patch clamp system with success rates >40% for GΩ seals. We demonstrate this method using hERG expressed in HEK cells, as well as Na1.5, Na1.7, and K3.1 expressed in CHO cells. We describe the advantages and disadvantages of using fluoride and provide examples of where fluoride can be used, where caution should be exerted and where fluoride-free solutions provide an advantage over fluoride-containing solutions.
几十年来，氟化物一直被用于手动和自动膜片钳实验的内部记录溶液中，因为它有助于提高封接电阻并促进更持久的记录。在手动膜片钳中，氟化物已被用于记录电压门控钠（Na）通道，其中封接电阻和接入电阻对于良好的电压控制至关重要。在自动膜片钳中，从膜片钳芯片下方施加吸力，以将细胞吸引到孔中并获得良好的封接。由于自动膜片钳的孔径不能像手动膜片钳中的膜片钳吸管那样移动以改善封接，自动膜片钳制造商使用内部氟化物来提高获得千兆欧封接的成功率。然而，内部氟化物会影响激活和失活的电压依赖性，以及影响内部第二信使系统，因此，希望能够选择使用无氟的生理内部溶液进行实验。我们已经开发出一种方法，可在基于384孔的自动膜片钳系统上进行高通量无氟记录，千兆欧封接的成功率>40%。我们使用在HEK细胞中表达的hERG以及在CHO细胞中表达的Na1.5、Na1.7和K3.1来演示此方法。我们描述了使用氟化物的优缺点，并举例说明了氟化物可用于何处、应谨慎使用何处以及无氟溶液相对于含氟溶液的优势所在。
