Perkin elmer 1420 multilabel counter victor3

PU-MC composites were digested with 0.5 mg/mL proteinase K at 56 °C overnight and used for DNA and glycosaminoglycan (GAG) measurement. DNA concentrations were determined with the Hoechst method using calf DNA as a standard. Fluorescence intensity was measured with an HTS 7000 Perkin Elmer Bio Assay Reader (Perkin Elmer, Milan, Italy). The amount of glycosaminoglycan (GAG) was determined by the dimethylmethylene blue dye method, using bovine chondroitin sulphate as standard. The total GAG content of the culture media, collected every 3 days, was also measured to assess the release of matrix molecules from the sample into the media. Absorbance was measured with a Victor3 Perkin Elmer 1420 multilabel counter (Perkin Elmer, Milan, Italy). GAG values were normalized to the DNA content.

In this experiment Pseudomonas aeruginosa PAO1 was used as a reference strain. Bacteria were grown in Mueller-Hinton medium (solid and liquid) at 37°C. Cells stored at– 80°C were spread onto Mueller-Hinton solid medium and grown 16 h at 37°C; colonies were transferred in liquid medium and incubated overnight at 37°C under constant shaking. The next day cells were diluted in fresh Mueller-Hinton liquid medium and grown at 37°C with shaking up to 0.2 O.D._600nm, then the cells were inoculated in a 96-well microplate in the presence or absence of rPON2 (50 and 100 μg/ml), PON1 (100 μg/ml), and incubated at 37°C in a humid atmosphere for 48 h. PAO1 was used as control in the presence or not of the enzyme buffer (20 mM Hepes pH 8.5 containing 0.5 mM Ca⁺⁺). After incubation, the medium was removed from the wells, and wells were washed three times with distilled water. The crystal violet staining was performed essentially as described by Nagant and colleagues [31 (link)]; the absorbance of each well was read at 540 nm in a Victor3 PerkinElmer 1420 Multilabel counter (PerkinElmer, Waltham, MA, USA). Each condition was replicated in six different wells and the experiment was carried out twice.

The cell apoptosis of treated and non-treated cells was detected using the CaspACEAssay^TM System according to the manufacturer’s protocols (Promega). Wells in duplicates containing blank (no cell extract), negative control (extract from untreated cells), induced apoptosis (extracts from PIN1 inhibitor-treated cells) and cells treated with caspase inhibitor (extracts from PIN1 inhibitor- and Z-VAD-FMK the caspase inhibitor-treated cells) were included in the experiments. The experiments were performed in triplicate and the absorbance of the colors developed was measured at a wavelength of 405 nm by a Perkin Elmer 1420 Multilabel Counter Victor 3 (Perkin Elmer). The caspase-specific activity was calculated according to the manufacturer’s guidelines.