Synergy ht microplate reader spectrophotometer

The mitochondrial activity of the cells and therefore the cell viability was evaluated by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [29 (link)]. At the time of the analyses, the media was discarded from each well and replaced with fresh medium supplemented with MTT at a final concentration of 100 µg·mL⁻¹. ARPE19 cells were incubated for 3 h at 37 °C in 5% CO₂ atmosphere and 400 μL of DMSO were then added to each well and mixed thoroughly by shaking to solubilize the purple formazan crystals formed from MTT reduction after incubation. The absorbance was read on a multiwell scanning microplate reader (BioTek Synergy HT MicroPlate Reader Spectrophotometer) at 570 nm using the DMSO as blank. The optical density in the control group (untreated cells) was considered as 100% viability. The relative cell viability (%) was calculated as follow:
Each experiment was performed at least five times in triplicate.

The hemolytic activity of both compounds was evaluated as described by Ghosh et al. [36 (link)]. Briefly, 4 mL of freshly drawn, heparinized human blood was diluted with 25 mL of phosphate buffered saline (PBS), pH 7.4. After washing three times in 25 mL of PBS, the pellet was resuspended in PBS to 20 vol %. A 100 μL amount of erythrocyte suspension was added to 100 μL of different concentrations of compounds 1 and 2, respectively. PBS and 0.2% Triton X-100 were used as the negative and positive control, respectively. Each condition was tested in triplicate. After 1 h of incubation at 37 °C each well was centrifuged at 1200 × g for 15 min, the supernatant was diluted 1:3 in PBS and transferred to a new plate. The OD₃₅₀ was determined using the Synergy HT microplate reader spectrophotometer (BioTek, Winooski, VT, USA). The hemolysis (%) was determined as follows:
where A is the absorbance of the test well, A₀ the absorbance of the negative control, and A_total the absorbance of the positive control; the mean value of three replicates was recorded.

The role of the sub-inhibitory concentrations of the tested antibiotics was verified even in biofilm and c-di-GMP production, using a P. aeruginosa PAO1-N strain, carrying a plasmid encoding the GFP protein under the influence of the cdrA promoter and responding to c-di-GMP levels. P. aeruginosa biofilms were developed in 96-well flat bottom dark plates in MH broth, with/without 1/4× MIC of each antibiotic. When testing the combinations ceftazidime/avibactam and ceftolozane/tazobactam, the β-lactamase inhibitor concentration was set at 4 µg/mL.
Biofilm production was then quantified as previously described [11 (link)]; for the c-di-GMP detection, the GFP fluorescence was measured in P. aeruginosa biofilms. After removing the planktonic phase and washing the plate wells with water, biofilms were mechanically detached and resuspended in PBS. The plate was then read using a Synergy HT MicroPlate Reader Spectrophotometer (BioTek, Winooski, VT, USA), measuring both biofilms optical density (OD) at 550 nm and fluorescence (excitation at 485 nm, emission at 535 nm). The fluorescent signal was normalized by calculating the Fluorescence/OD₅₅₀ ratio.