F 2000 fluorescence spectrophotometer

Fibril formation was monitored using standard Thioflavin T fluorescence assays^{55 (link)} using a Hitachi F-2000 fluorescence spectrophotometer (λ_ex 446 nm, λ_em = 490 nm, averaged over 10 s, three measurements per sample. To estimate lag periods, measured values were fitted to the equation of a stretched exponential equation, $$T=T_0+\left(T_{\mathit{eq}}-T_0\right)\left(1-e^{-k{t}^n}\right)$$ were T₀ = fluorescence at time 0, T_eq = fluorescence at infinite time, t = time, k is a rate constant (s⁻¹) and n is a dimensionless parameter. Fibrils were examined by transmission electron microscopy by previously described procedures^{53 (link)}.

All sorption experiments were conducted at room temperature in batch mode with 0.1 g of cross-linked polymer and 10 mL of fluoroquinolone (i.e., CIP or OFL) water solution (concentration range from 0.01 to 0.2 mg/L). The experiments were carried out in Falcon tubes, and the suspensions were mixed in a tube rotator (VWR) for 3 h. Then, the quinolone solution was separated by centrifugation at 4000 rpm, and its concentration was quantified with an F2000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) by interpolating the emission at 452 nm (λex 273 nm) in a calibration curve.
The ability of cross-linked polymers to sorb CIP and OFL was evaluated by the sorption coefficient, Kd, which is defined as the ratio between the concentration of the fluoroquinolone in solution (Ce) and in the polymer (qe) and is estimated as the slope of the plot qe (mg/kg) vs. Ce (mg/L) at equilibrium [21 (link)]

Lipid peroxidation was assessed by determining the level of thiobarituric acid-reactive substances (TBARS) as previously described [13 ]. Briefly, 200 μL liver homogenate was mixed with 250 μL of 25% (w/v) trichloroacetic acid, and then, the mixture was centrifuged at 10,000 x g for 30 min at 10^oC. The resulting supernatant was collected and reacted with an equal volume of 1% thiobarbituric acid (TBA) for 40 min at 95°C in the dark for the formation of TBARS. After cooled at room temperature for 15 min, quantitation of TBARS was performed by using a Hitachi F2000 fluorescence spectrophotometer (excitation at 532 nm and emission at 600 nm) and 1, 1, 3, 3-tetraethoxypropane (TEP) standard curve (0–50 nM, r² = 0.9951).

An amount of 50 mg of pSt was incubated in a Falcon tube with 9 mL of a 20 mg/mL CIP solution supplemented with 1 mL of buffering solution (sodium acetate pH 4.6 or 6, HEPES pH 7 or 8, Tris-HCl pH 8 or carbonate pH 9.5) to provide a buffer concentration of 10 mM, 25 mM or 50 mM and pH values of 4.6, 6.0, 7.0, 8.0, and 9.5. Suspensions were mixed in a tube rotator (VWR, Radnor, PA, USA) for 3 h. Then, the quinolone solution was separated by centrifugation at 4000 rpm, and its concentration was quantified with an F2000 fluorescence spectrophotometer (Hitachi) by interpolating the emission at 452 nm (λex 273 nm) in a calibration curve.
The effect of high concentrations of NaCl was evaluated on 100 mg of pSt and 0.2 mg/mL CIP in either water or 10 mM HEPES pH 8. The solutions were supplemented with NaCl to a final concentration of 4‰, 14‰, and 24‰ (brackish water); 35‰ (seawater); and 50‰ (brine), and the experiment proceeded as described above.

Mitochondrial activity in collagen sponges was evaluated by an MTT (3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay at D28. 400 µl of culture medium and 100 µl of MTT solution (5 mg/ml MTT in PBS) were added per collagen sponge and plates were incubated in the dark at 37°C under 5% CO₂ for 4 hours. The intense purple coloured formazan derivative formed during active cell metabolism, was eluted and dissolved in a solution containing sodium dodecyl sulfate with dimethylformamide. The absorbance was measured at 580 nm. Mitochondrial activity results were normalized by DNA quantisation in each sponge. To evaluate total DNA quantity, Hoechst 33258 dye (Molecular Probes) was used. Working assay solution was prepared fresh prior to each assay by mixing 1 µl of concentrated dye stock solution (0,1 mg/ml) for every 1 ml of assay buffer required resulted in a final Hoechst 33258 concentration of 0,1 µg/ml. Cells were lysed by freeze-thawing in 100 µl of a buffer containing 10 mM Tris, 1 mM EDTA and 0,1 M NaCl, pH 7,4. Then, 2 ml of working Hoechst solution were added in each sample. Results were extrapolated from a standard curve based on calf thymus DNA (D-3664, Sigma, France) ranging from 0 to 0.5 µg/ml. Samples were measured fluorometrically (F2000 Fluorescence Spectrophotometer, Hitachi) at excitation wavelength of 348 nm and emission wavelength of 456 nm.

The cells were labelled for 10 min at 37°C with 5 µM 6-carboxyfluorescein diacetate (6-CFDA, Molecular Probe, Invitrogen) cell-permeable esterase substrate and exposed to plasma. The cells were then seeded (8×10⁴ cells/ml) in DMEM supplemented with 0.2% vol/vol FBS into trans-well inserts (8 µm pore size, Corning, the Netherlands), held in 12-well tissue culture plates containing complete culture medium and cultured for 72 hrs. The bottom side of the trans-well inserts was washed twice and the cells were harvested using cell scrapers. The cell membrane was dissolved in PBS containing 0.5% vol/vol Triton X-100. The samples were then centrifuged at 4°C 13,000 rpm for 10 min. Fluorescence was detected in the supernatant at excitation and emission wavelengths of 485 and 530 nm, respectively (Hitachi F2000 Fluorescence Spectrophotometer, Japan).