Spectramax i3 plate

To investigate the safety of phage isolate CSP1 on human cells, we utilized the Cell Counting Kit-8 (CCK-8) (GLPBIOGK10001) to assess the cytotoxic effects of CSP1 on human embryonic kidney cells 293 (HEK293 T) and lung adenocarcinoma cells (A549) in vitro. HEK293T and A549 cells were seeded in a 96-well plate at a density of 5,000 cells/well in 100 μL. Each well received 10 μL of the bacteriophage CSP1 at initial concentrations of 3×10¹² PFU/mL, followed by a series of tenfold dilutions, resulting in phage concentrations of 3×10¹⁰, 3×10⁹, 3×10⁸, and 3×10⁷ PFU/well. For the control setup, wells 1 and 2 each held 100 μL of cell suspension; well 1 was augmented with 10 μL of LB medium, while well 2 received 10 μL of standard cell culture medium. Following treatment, the plates were incubated in a cell culture incubator for 12 or 24 hours, after which CCK-8 was added to each well except for three replicate wells per experimental group, which did not receive CCK-8 to allow for the determination of phage titer in the phage-cell co-culture. The incubation was continued at 37°C for an additional hour. The optical density at 420 nm was measured using the Molecular Devices SpectraMax®i3 plate reader, and data were analyzed in triplicate for each condition. Additionally, the titer of bacteriophage CSP1 without CCK-8 in the experimental groups was determined.