Glass chamber slides

Fibroblasts were grown on glass chamber slides (Corning) for 24 h. Afterwards cells were FCS starved. Cells were incubated with different amounts or no FCS (0, 0.5, 1, 2, 4, and 10%), with or without 100 µM AA. Supernatants were frozen at −80 °C until further use. Cells were stained with Sirius Red (SR) according to [33 (link)]. Briefly, cells were fixed with methanol ON at −20 °C and carefully washed with PBS and stained with 1% picro-Sirius red solution (Sigma) for 1h at RT. Cells were washed with 0.1% acidic acid, treated with ethanol followed by xylene and mounted with Entellan (Merck, Darmstadt, Germany). Image acquisition was conducted using a Nikon C2 Eclipse microscope.

Glass chamber slides (Corning) coated with either 10 or 20 mg/ml of Lp‐BS or acrylic discs coated with 50, 0.097, and 0.048 mg/ml rhamnolipid were prepared and then incubated with bacterial suspension (absorbance₆₀₀ = 0.6 in PBS) for 2 h at 4°C. Bacterial suspension was removed and the wells/acrylic discs washed with 200 μl of PBS. Glass‐attached bacterial cells were harvested using a cell scraper into 1 ml of sterile PBS and vortexed. Discs were placed in sterile plastic bijoux bottles containing 5 ml of PBS and vortexed at 2500 rev/min for 30 s to detach bacteria. Ten‐fold serial dilutions (up to 10⁻⁴) were prepared in sterile PBS and 50 μl spirally plated onto FAA plates (spiral plater; Don Whitley Scientific), which were then incubated in 5% CO₂ at 37°C for 24–48 h until colonies were visible to count. Results were expressed as log₁₀ CFU per ml from three independent experiments.