| Assay Method Information | |
| | Quorum sensing assay |
| Description: | 2.1 The wells of a 12-well plate were marked as initial concentration group, 2, 4, 8, 16, 32, 64, 128, and 256 fold diluted group, DMSO group, and a blank control group from left to right and from up to bottom, respectively.2.2 The monoclonal C. violaceum CV026 grew on LB solid plate was cultured to exponential phase in a 5 ml fresh LB liquid culture medium, and 50 μl was then taken for seeding in a 5 ml LB culture medium, and cultured until the optical density OD value was about 1.0. The culture was then mixed homogeneously with LB culture medium at a ratio of 1:9 (at an OD of about 0.15), and added to a 12-well plate in an amount of 2 ml/well.2.3 The compounds having quorum sensing inhibitory activity in preliminarily screening were dissolved in DMSO (at a concentration of 0.065M), respectively, and then 10 μl was taken into 10 μl DMSO solution for 2-fold dilution, and so on. Each compound was gradiently diluted to a highest fold of 256 (gradient dilution of 2, 4, 8, 16, 32, 64, 128, 256 fold).2.4 To each well, 15 μl 1000-fold diluted inducer C6-HSL (initial concentration of 0.125M) and 8 μl compound solution at each diluted gradient was added; to the DMSO group, 8 μl DMSO was added; to the blank control group, 8 μl LB culture medium was added. Finally, it was ensured that 2 ml culture in each of the 12-well plate was mixed homogeneously.2.5 The 12-well plate was placed in a 30° C. shaker at 130 rpm and cultured for 10-12 h.2.6 After the culture was finished, 1 ml culture was taken from each well and put in a 1.5 ml EP tube and then centrifuged at 12000 rpm for 10 mins. The supernatant of the culture was sucked out, and 500 μl DMSO was added to each EP tube to dissolve the purple pigment in the culture. After the pigment was completely dissolved, centrifugation was performed at 12000 rpm for 10 mins. 200 μl supernatant pigment was put in a 96-well culture plate, and measured for absorbance value at 585 nm by a Microplate Reader. The absorbance value and the corresponding concentration were plotted to get the IC50 value. |
| Affinity data for this assay | |
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