FRET-based αSynuclein Oligomerization Assay

HEK293 and SH-SY5Y cells (ATCC) were cultured in phenol red-free Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 2 mM L-Glutamine (Invitrogen), heat-inactivated 10% fetal bovine serum (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). Cell cultures were maintained in an incubator with 5% CO₂ (Forma Series II Water Jacket CO₂ Incubator, Thermo Scientific) at 37 °C. The inter-protomeric (oligomer) and intra-protomeric (oligomer and conformation) αSN FRET biosensors were generated by transiently transfecting HEK293 cells using Lipofectamine 3000 (Invitrogen) with GFP-αSN and αSN-RFP (1:8 DNA plasmid concentration ratio) or GFP-αSN-RFP plasmid, respectively. The effectiveness of HEK293 cells transfected with FRET constructs as an HTS platform has been demonstrated in our previous work^{46 (link)–52 ,66} .