Cytotoxicity Assessment of Lipofectin and Phages

Lipofectin (Thermo Fisher Scientific; Cat No.: 18292-037) and phage particles (T7-WT, phage 54 and phage 74) were prepared as described previously (24 (link)). In vitro cytotoxicity of Lipofectin and phage particles was evaluated using the Cell Counting Kit-8 (CCK-8) reagent (Dojindo Laboratories, Japan) according to the manufacturer’s instructions. Based on the protocol described by Zhao et al. (26 (link)), HEK293T cells were seeded in a 96 well plate at a density of 1×10⁴ cells per well and incubated at 37°C in a 5% CO₂ humidified atmosphere for 24 h. Subsequently, the medium was replaced by fresh Dulbecco’s Modified Eagle Medium (DMEM) mixed with 0, 5, 10, 15 and 20 μL Lipofectin to a final volume of 100 μL or by 100 μL fresh DMEM containing a final concentration of 0, 5×10⁸, 1×10⁹, 5×10⁹ and 1×10¹⁰ pfu/mL phage particles. After a 4 h incubation, Lipofectin and phages were removed by a single wash, and cells were cultured for an additional 24-72 h. Thereafter, 10 μL of CCK-8 reagent was added and incubated for another 4 h. The absorbance at 450 nm was read with a microplate reader (iMark microplate reader, BIO-RAD). Cells without any treatment were set as 100% viability. All measurements were performed in triplicate.