Automation is used for many applications to reduce variation caused by manual handling and to obtain reproducible results in high-throughput assays. High-throughput applications, such as knockdown studies or target screenings, often include cell transfection. In this study, X-tremeGENE HP and X-tremeGENE 9 Transfection Reagents (Roche) were tested for their compatibility with an automated transfection workstation, the Biomek FX (Beckman Coulter).
Material and Methods
Cell seeding and transfection were performed using the Biomek FX automated workstation. For the transfection, X-tremeGENE HP and X-tremeGENE 9 reagents (Roche) were prediluted in Opti-MEM (dilution factor: 1:50 for X-tremeGENE HP reagent, 1:33 for X-tremeGENE 9 reagent). The EGFP-PC3.1 plasmid was prediluted in Opti-MEM (1 µg/100 µL). The transfection-complex was formed by pipetting the same amount of plasmid dilution into the dilution of transfection reagent, followed by mixing and incubation for 30 min.
Cell Culture and Reverse Transfection
HeLa cells (ATCC® CCL-2™) were cultured in MEM with supplements at +37°C in a 5% CO2 humidified atmosphere in T75 flasks until a confluence of about 70% was reached (Figure 1). Cells were harvested by trypsinization shortly before transfection, washed and resuspended in fresh medium (1.2x105 cells/mL). Reverse transfection was performed by pipetting 100 µL cell suspension to each well already containing the transfection complex.