Optimising electroporation protocols for Trypanosoma brucei using the Amaxa 4D-nucleofector system.

Stable transfection of Trypanosoma brucei remains a cornerstone for functional genetic studies in this model parasite. Although the Amaxa Nucleofector II system dramatically improved transfection efficiency in both monomorphic and pleomorphic bloodstream forms, the more recent 4D Nucleofector platform offers enhanced programmability and buffer flexibility that have yet to be systematically evaluated for T. brucei. Here, we benchmark a range of 4D Nucleofector programs to determine optimal parameters for transfection efficiency, cell viability, and reproducibility in bloodstream forms. Using a CRISPR/Cas9 expressing cell line, we compare stable transfection efficiencies across programs. We further demonstrate the advantages of the 16-well Nucleocuvette™ Strip format, enabling simultaneous processing of multiple experimental conditions in 20 µL reactions, reducing DNA, cell, and reagent requirements while increasing experimental throughput. Our results provide a standardized framework for future genetic manipulation of T. brucei using the 4D-Nucleofector X Unit, facilitating robust and reproducible transfection across life-cycle stages and strains.