Cell culture assay for sonicate cytotoxicity.

Overnight cultures of E. coli strains were pelleted by centrifugation at 13,500 × g for 5 min. The pelleted cells were washed in 1 mL of PBS and pelleted again by centrifugation at 13,500 × g for 5 min. Pellets were resuspended in 2 mL of PBS and then sonicated on ice (amplitude, 35; power, 7 W; 30-s intervals) for a total of 5 min with 1-min breaks between intervals. Sonicated samples were centrifuged at 13,500 × g for 10 min at 4 °C to remove large debris. Supernatant was collected and then filter-sterilized through 0.2 μm filters. Total protein was quantified by using the BCA assay (Thermo Fisher Scientific). HeLa cells (5 × 10³ per well) were seeded onto 96-well cell culture plates and incubated at 37 °C with 5% CO₂ for 24 h. Cells were treated with 1 or 40 μg/mL total protein of crude bacterial sonicate for 72 h. Cells were stained and microscopically analyzed for confluence and morphologic changes as described earlier.