Fluoride-free high-throughput automated patch clamp
For decades, the addition of fluoride to internal solutions in patch clamp assays, both manual and automated, has been a standard practice to enhance seal resistance and extend the duration of recordings. In manual patch clamping, fluoride has been routinely used when studying voltage-gated sodium (NaV) channels, ensuring high seal resistance, crucial for accurate voltage control. In automated patch clamping (APC), where cells are drawn towards a hole on the patch clamp chip to secure a seal, fluoride in the internal solution is used to increase the likelihood of achieving high-quality GΩ seals.
However, it should be noted that internal fluoride can alter the voltage dependence of activation and inactivation of certain channels and interfere with internal second messenger pathways. Consequently, there is increasing demand for the ability to conduct experiments with a physiological, fluoride-free internal solution to avoid these alterations and more closely replicate the natural cellular environment.
The study “There is no F in APC…” introduces a novel approach to conducting high-throughput APC experiments without fluoride, demonstrating its viability and effectiveness across several ion channel types, including hERG, NaV1.5, NaV1.7, and KCa3.1, expressed in different cell lines. The authors used a special type of fluoride-free chips (NPC-384FF) to systematically explore the implications of omitting fluoride from APC experiments, focusing on seal resistance, success rates, and the biophysical properties of different ion channels. By employing a 384-well-based APC platform, the SyncroPatch 384, they achieved success rates exceeding 40% for gigaohm seals without fluoride, challenging the conventional belief that fluoride is indispensable for high-quality, high-throughput APC recordings.
Importantly, the proposed approach also worked on stem cell-derived cardiomyocytes. In preliminary recordings, the authors successfully recorded CaV currents in hiPSC-CMs using physiological solutions, suggesting that this approach is not limited to standard cell lines but could be adopted for a wide range of cell types and experiments.
In conclusion, this study represents a significant advancement in APC technology by showcasing the feasibility of high-throughput fluoride-free experiments, making APC data more comparable to traditional manual patch clamp results in physiological solutions. Ultimately, this will enhance ion channel characterization and drug testing across various cell types, including cell lines, induced pluripotent stem cells, and primary cells.
Find the original article here: There is no F in APC: Using physiological fluoride-free solutions for high throughput automated patch clamp experiments
Learn more about the automated patch clamp.