Transfection methods for high-throughput cellular assays of voltage-gated calcium and sodium channels involved in pain.
Category:- Journal; Year:- 2021
Discipline:- Pharmacy Discipline
School:- Life Science School
Chemical transfection is broadly used to transiently transfect mammalian cells, although often associated with cellular stress and membrane instability, which imposes challenges for most cellular assays, including high-throughput (HT) assays. In the current study, we compared the effectiveness of calcium phosphate, FuGENE and Lipofectamine 3000 to transiently express two key voltage-gated ion channels critical in pain pathways, Ca V 2.2 and Na V 1.7. The expression and function of these channels were validated using two HT platforms, the Fluorescence Imaging Plate Reader FLIPR Tetra and the automated patch clamp QPatch 16X. We found that all transfection methods tested demonstrated similar effectiveness when applied to FLIPR Tetra assays. Lipofectamine 3000-mediated transfec-tion produced the largest peak currents for automated patch clamp QPatch assays. However , the FuGENE-mediated transfection was the most effective for QPatch assays as indicated by the superior number of cells displaying GΩ seal formation in whole-cell patch clamp configuration, medium to large peak currents, and higher rates of accomplished assays for both Ca V 2.2 and Na V 1.7 channels. Our findings can facilitate the development of HT automated patch clamp assays for the discovery and characterization of novel analgesics and modulators of pain pathways, as well as assisting studies examining the pharma-cology of mutated channels.