✍🏼 Mohammad-Reza Ghovanloo, PhD, Philip R. Effraim, MD, PhD, Sidharth Tyagi, MD, PhD, Alecia M. Aldrich, BSc, Xiaoyang Cheng, PhD , Jun-Hui Yuan, MD, PhD, Betsy R. Schulman, PhD, Deborah S. Jacobs, MD, Sulayman D. Dib-Hajj, PhD , and Stephen G. Waxman, MD, PhD

🏠 Department of Neurology, Yale University School of Medicine, New Haven, CT, USA

📑 Neurology Genetics (2024)

Abstract
Despite extensive efforts, the mechanisms underlying pain after axonal injury remain incompletely understood. Pain following corneal refractive surgery offers a valuable human model for investigating trigeminal axonal injury because laser-assisted in situ keratomileusis (LASIK) severs axons of trigeminal ganglion neurons innervating the cornea. While the majority of patients are pain-free shortly after surgery, a minority endure persistent postoperative ocular pain. Through genomic analysis of patients experiencing persistent postoperative ocular pain, we identified rare variants in genes encoding ion channels and receptors, including TRPM8, which codes for the menthol-sensitive and cold-sensing transient receptor potential cation channel.

How the WOLF was used in this study
The WOLF G2 cell sorter was used to enrich for HEK-293 cells successfully expressing wild-type or mutant TRPM8 channels prior to functional analysis. Following transient transfection with TRPM8 constructs tagged with 2A-GFP, cells were subjected to fluorescence-activated cell sorting on the WOLF G2 to isolate GFP-positive populations, ensuring a highly purified and uniform set of transfected cells. At least 500,000 GFP⁺ cells were collected per condition, and post-sort inspection confirmed >95% fluorescence purity. This sorting step was critical for downstream automated patch-clamp electrophysiology, as it minimized variability from untransfected cells and ensured that observed biophysical and pharmacologic differences could be directly attributed to the TRPM8 variants under investigation.