Fluorstar optima

E9 cells were seeded at 2000/well in 96 well plates (Corning, USA) containing 100 µl of supplemented media as described. After 24 h, media was discharged and changed with new media added with different concentrations of CAR agonist (TCPOBOP) or CAR inverse agonist (androstenol) from 10⁻⁴ µM to 10 µM. Forty-eight hours later, 11 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT - 5 mg/mL) was added to each well and formazan crystals were produced over a 2 h incubation period. Medium was removed from each well and 100 µl of 0.4N HCl in isopropilic alcohol were added to dissolve crystals. Optical density at 540 nm was measured in a Fluorstar Optima (BMG Labtech, Germany).

The insoluble and soluble EPS were quantified as previously performed.⁵ Total carbohydrates were measured using the phenol-sulphuric acid colorimetric assay under absorbance of 490 nm using a microplate reader (Fluorstar Optima- BMG Labtech; Ortenberg, Baden-Württemberg, Germany).²⁶ The values for both EPS were expressed as μg EPS/mg (biofilm).⁵

The serum collagen concentration was determined using the Soluble Collagen Assay Kit (ab241015, abcam). The fluorescence (Ex/Em =  340/460 nm) was measured in endpoint mode by a fluorometer (FluorStar Optima, BMG Labtech).

Total amount of ROS was measured by the oxidation of the DCFDA fluorochrome (Sigma 35848), which is esterified by cellular esterase’s to DCFH, and this is oxidized to DCF in the presence of reactive species [49 (link)]. The subsequent fluorescence was measured using the FLUOrstar OPTIMA (BMG Labtech, Ortenberg, Germany) and was presented as nmol/mg of DCF.

CD73 enzymatic activity in CAF-S1 primary cell lines was assessed using AMP GLO^TM luminescence assay (Promega, Madison, WI, USA, #V5011) allowing the measurement of exogenous AMP hydrolysis. Harvested CAF-S1 were suspended in supplemented DMEM at the concentration of 4 × 10⁶ cells/mL. A total of 25 µL of CAF-S1 was plated in 96 well-plate U-bottom (Falcon, Corning, NY, USA, #353077) alone, in presence of anti-CD73 or isotype control antibody at 10 µg/mL, or the pharmacological inhibitor, APCP, at 100 µM. Cells were incubated 1 h at 4 °C. A total of 25 µL of AMP 50 µM diluted in supplemented DMEM was added for 1 h at 4 °C. After a rapid centrifuge, 25 µL of supernatants were then moved in 96-well white microplate (Grainer, #655083) to quantify the residual AMP with AMP-Glo^TM luminescence assay according to manufacturer’s instructions. Emitted light was measured on plate reader (Fluorstar Optima, BMG Labtech). The relative luminescence units (RLU) were proportional to the residual AMP within supernatants. The percentages of residual AMP were calculated as follow: %residual AMP = relative luminescence unit (CAF-S1 untreated or treated with anti-CD73, isotype CTL antibody or APCP)/relative luminescence unit (AMP alone) × 100. %degraded AMP = 100 − %residual AMP.

The epigenetic probes (Cayman Chemical, Ann Arbor, MI, USA) were dissolved in dimethylsulfoxide (DMSO) to a concentration of 20 mM. A549 cells were seeded at 5000/well in 96-well plates (Corning, NY, USA) containing 100 µL of supplemented media, as described previously. After 24 h, the medium was replaced with fresh culture medium containing different concentrations of epigenetic probes, ranging from 10 µM to 13.72 nM. epigenetic probes were added in six replicates per concentration and the experiments were performed in triplicate. After 72 h, 10 µL of 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide (MTT, 5 mg/mL) was added to each well and formazan crystals were produced over a 2 h incubation period. One hundred microliters of DMSO were added to dissolve the crystals. The optical density at 540 nm was measured using Fluorstar Optima (BMG Labtech, Ortenberg, Germany). The concentration of the compound corresponding to the IC₅₀ was calculated using a nonlinear regression test performed in GraphPad Prism (version 6.00 for Windows, GraphPad Software, USA).