Cayman camp eia assay kit

Intracellular cAMP concentrations were measured using the Cayman cAMP EIA assay kit. P. aeruginosa cultures of indicated strains were incubated 15–16h with aeration at 37°C in LB, diluted 1:100 into fresh medium the next morning, and grown with aeration to OD₆₀₀ 0.4–0.6. Cells were harvested by centrifugation (20,000 xg for 2 min at 4°C) and washed twice with 1 ml cold 0.9M NaCl. Pellets were suspended in 150 μl 0.1N HCl and incubated on ice for 10 min with occasional vortexing. The lysates were centrifuged at 20,000 xg for 5 min at 4°C and supernatants were used to measure intracellular cAMP concentrations via the Cayman cAMP EIA assay kit, following the manufacturer’s protocol for sample acetylation.