Falcon 353072

Human adenosine A₃ receptor functional experiments were carried out in CHO-A₃#18 cell line. The day before the assay, the cells were seeded on the 96-well culture plate (Falcon 353072, Corning, Glendale, AZ, USA). The cells are washed with wash buffer (Dulbecco’s modified eagle’s medium nutrient mixture F-12 ham (Sigma D8062), 25 mM Hepes; pH = 7.4). Wash buffer is replaced by incubation buffer (Dulbecco’s modified eagle’s medium nutrient mixture F-12 ham (Sigma D8062), 25 mM Hepes, 30 μM Rolipram (Sigma R6520); pH = 7.4). Test compounds and MRS1220 as reference compound (Sigma M228) are added and the cells incubated at 37 °C for 15 min. After, 0.1 μM of 5′-N-ethylcarboxamido-adenosine (NECA) (Sigma E2387) is added and the cells incubated at 37 °C for 10 min. Forskolin (Sigma F3917, Sigma-Aldrich, St. Louis, MO, USA) is added and incubated at 37 °C for 5 min. After incubation, the amount of cAMP is determined using a cAMP Biotrak Enzymeimmunoassay (EIA) System Kit (GE Healthcare RPN225, GE Healthcare, Chicago, IL, USA).

Biofilm formation assays were performed following the methodology previously described (Domenech et al., 2015 (link)) and it was determined by the ability of cells to adhere to the walls and base of 96-well, flat-bottomed polystyrene microtiter dishes (Falcon 353072, Corning Incorporated, New York, NY, United States). Unless stated otherwise, cells were grown in C+Y medium to an A₅₅₀ of ≈0.5–0.6, sedimented by centrifugation, resuspended in an equal volume of the indicated pre-warmed medium, diluted 1/100, and then dispensed 200 μl per well. After 5 h of incubation at 34°C, the A₅₉₅ was determined using Tecan Infinite F200 (Tecan Group Ltd., Switzerland). The biofilm formed was stained with 0.2% crystal violet (Sigma-Aldrich) and rinsed three times with distilled water to remove non-adherent bacteria. Biofilm formation was quantified solubilizing the biofilm in 95% ethanol (200 μl per well) and measuring the A₅₉₅.