Elastin congo red conjugate

In the cell-free culture, supernatants of each P. aeruginosa strain cultivated in the presence and absence of EOs and limonene, pyocyanin, and elastase activities were quantified as described by Díaz et al. [30 (link)]. The elastolytic activity in the supernatants was evaluated using the elastin–Congo red conjugate (Sigma–Aldrich, St. Louis, MO, USA) at 495 nm. At the same time, the pyocyanin activity was determined using the chloroform–HCl extraction method and was quantified via absorbance measurements at 520 nm. DMSO-treated cultures were used as controls, and each test was assessed for statistical significance (n = 3).

The effect of ACP treatment on P. aeruginosa elastolytic activity (Las B), controlled by the las QS system, was determined using elastin-Congo red conjugate (Sigma Aldrich, Ireland), as the most specified substrate for elastase, following the procedure described by [29 (link)]. Either untreated 0 h and 24 h controls or ACP treated bacterial suspensions were centrifuged for 10 min at 10,000 rpm. Elastin-Congo red (2.5 mg) was suspended in 500 μl of 0.2 M Tris buffer (pH 8.8) in an eppendorf, vortexed, and mixed with 500 μl of P. aeruginosa supernatant. The resulting solution was incubated at 37°C for 24 h. After incubation, the samples were vortexed and centrifuged at 13,000 rpm for 10 min. The supernatant was dispensed into the wells of the 96 well microtiter plate and the absorbance of released Congo red was measured at 495 nm. An average absorbance, corrected by the mean absorbance obtained from the corresponding mixture incubated in the absence of elastin-Congo red, represents P. aeruginosa enzymatic elastase activity. Experiments were repeated at least five times.