A Novel, Fully Automated, and Reagent-Agnostic Transient Transfection Protocol

Transfection is a potent technique to introduce foreign nucleic acids into eukaryotic cells. The capacity of the technique to alter the genetic content of host cells means it is useful for a wide range of applications, including the study of typical cellular processes, disease molecular mechanisms, and gene therapy effects. Here, we discuss a highly reliable and fully automated transient transfection protocol that utilizes an open-source liquid handler and accompanying HEPA Module. Two commonly used transfection reagents are employed to study the transfection efficiency in two cell lines with a GFP plasmid construct. The detailed method of the protocol, image acquisition, and analysis for evaluating transfection efficacy is provided. With HeLa cells, the transfection efficiency of the reagents ranges from 40.92% to 73.26%, while with the difficult-to-transfect A549 cells, the transfection efficiency is between 42.15% and 54%. The efficiency achieved is comparable to similar experiments performed manually. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Seeding of adherent cells (A549 and HeLa) for transient transfection on a Costar 6-well plate using a liquid handler on Day 0 Basic Protocol 2: Transfection of the cell lines using the transfection reagents Lipofectamine 3000 and FuGENE HD on Day 1 Support Protocol: Image acquisition and semi-quantitative analysis of transfection after 24 hr to calculate the transfection efficiency.