Biomedical Engineering Department, Northwestern University, Evanston, IL 60208, USA.
IEEE Trans Biomed Eng. 2013 Jul;60(7):2042-51. doi: 10.1109/TBME.2013.2248084. Epub 2013 Feb 21.
During a conventional whole-cell patch clamp experiment, diffusible cytosolic ions or molecules absent in the pipette solution can become diluted by a factor of one million or more, leading to diminished current or fluorescent signals. Existing methods to prevent or limit cytosol diffusion include reducing the diameter of the pipette's orifice, adding cytosolic extract or physiological entities to the pipette solution, and using the perforated patch clamp configuration. The first method introduces measurement error in recorded signals from increased series resistance and the latter two are cumbersome to perform. In addition, most perforated patch configurations, prevent investigators from using test compounds in the pipette solution. We present a method to overcome these limitations by minimizing cytosol dilution using a novel pipette holder. Cell-attached configuration is obtained with the pipette filled with pipette solution. Most of the pipette solution is then replaced with mineral oil so that cytosol dilution can be minimized in whole-cell configuration. To accomplish this requires a suction line and two Ag/AgCl electrodes inside the pipette. Testing our novel pipette holder with Chinese Hamster Ovarian cells, we demonstrate cytosol dilution factors between 76 and 234. For large cells with somas greater than 40 μm, cytosol dilution factors of 10 or less are achievable.
在常规的全细胞膜片钳实验中，由于细胞浆中的可扩散离子或分子不存在于电极内液中，会被电极内液稀释一百万倍甚至更多，从而导致电流或荧光信号减弱。现有的防止或限制细胞质扩散的方法包括减小电极尖端的直径、向电极内液中添加细胞质提取物或生理实体以及使用穿孔膜片钳技术。前一种方法会因增加的串联电阻而在记录的信号中引入测量误差，而后两种方法则繁琐复杂。此外，大多数穿孔膜片钳技术的配置都阻止了研究人员在电极内液中使用测试化合物。我们提出了一种新的方法来克服这些限制，即通过使用一种新型的电极夹来最小化细胞质稀释。在充满电极内液的情况下，获得细胞贴附构型。然后将大部分电极内液替换为矿物油，从而在全细胞构型中最小化细胞质稀释。要实现这一点，需要在电极内设置一个吸管和两个 Ag/AgCl 电极。我们用中国仓鼠卵巢细胞对新型电极夹进行了测试，结果表明细胞质稀释系数在 76 到 234 之间。对于体积极大（超过 40μm）的细胞，可实现 10 倍或更低的细胞质稀释系数。
