Elastin congo red substrate

Pseudomonas aeruginosa cells were grown in LB at 37°C for 12 h, and cells were removed from 10 mL growth medium by centrifugation at 5,000 × g for 10 min at 4°C. LasA protease activity was determined by measuring the ability of P. aeruginosa culture supernatants to lyse boiled Staphylococcus aureus RN4200 cells, as described previously (Huang et al., 2016 (link)). A 30-mL overnight culture of S. aureus grown in tryptic soy broth was placed in a boiling water bath for 10 min and then centrifuged for 10 min at 10,000 × g. The resulting pellet was re-suspended in 10 mM Na₂HPO₄ (pH 7.5) and adjusted to an OD₆₀₀ of 0.9. A 100-μL aliquot of bacterial supernatant was then added to 900 μL of S. aureus suspension, and the OD₆₀₀ was determined after 5, 10, 15, 20, 25, 30, 35, 40, 45, 60, 75, 90, and 105 min. LasB protease activity in P. aeruginosa culture supernatants was determined using the elastin Congo red assay (Yuan et al., 2012 (link)). After a 3-h digestion period at 37°C (100 mM Tris, 1 mM CaCl [pH 7.5] with 20 mg of elastin Congo red substrate [Sigma] plus 100 μL of spent supernatant), suspensions were clarified by centrifugation (16,000 × g, 5 min, RT), and the absorbance at 495 nm was measured to determine the amount of liberated dye.

Cells were grown at 37 °C overnight in LB and collected by centrifugation at 16,000 × g for 1 min. Supernatant were passed through a 0.22 µm filter, and 100 µL of supernatant was mixed with 900 µL of 10 mM Na₂HPO₄ and 10 mg elastin-Congo red substrate (Sigma-Aldrich). After incubation for 2 h at 37 °C, and the samples were centrifuged at 16,000 × g for 10 min. The optical density of the supernatant was measured at OD₄₉₅. Each biological sample had three technical replicates.