Genios plus microplate reader

As previously described, collagen type-I (COLS) synthesis was investigated employing COL1A1-LV-tGFP, a GFP-based lentiviral vector (LV) driven by the human COL1A1 gene promoter [22 (link), 24 (link)]. A red fluorescence protein-based LV (EF1α-LV-FP602) was used to normalize the cell transduction efficiency [22 (link)]. This method allows us to perform the real-time assessment of multiple samples at the same time in a 96-well plate using a small amount of subject sera [22 (link)]. HPMECs were transduced with lentiviral particles obtained from the pCOL1A1-LV-tGFP and EFα-LV-FP602 lentivectors. Transduction efficiency was checked and confirmed under a fluorescence microscope. Treatment-induced variation of fluorescence was kinetically measured over a time-course of 12 hours using a Tecan GENios Plus microplate reader (Tecan, Switzerland). Excitation wavelengths used for fluorescence quantification were 485 nm and 535 nm, while emission wavelengths were 535 nm and 590 nm for pCOL1A1-LV-tGFP and EFα-LV-FP602, respectively. Data were normalized for transduction efficiency by reporting the ratio of pCOL1A1-LV-tGFP to EFα-LV-FP602 and expressed as a means ± SD of the relative fluorescence unit (RFU) values.

Intracellular ROS levels were determined by using the ROS molecular probe 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCF-DA) (Molecular Probe, Eugene, OR, USA), as previously described [23 (link),24 (link)]. Within the cell, esterases cleave the acetate groups on H₂DCF-DA, thus trapping the reduced form of the probe (H₂DCF). Intracellular ROS oxidize H₂DCF, yielding the fluorescent product, DCF. Cells were incubated for 3 h with the concentrations of extract indicated in the figure legends. After treatment, cells were incubated for 30 min with Hanks’ Balanced Salt Solution (HBSS) containing 5 µM H₂DCF-DA, then washed twice with HBSS and assessed for the fluorescence by using a Tecan GENios plus microplate reader (Tecan, Männedorf, CH, USA) in a light-protected condition. Treatment-induced variation of fluorescence was measured for 2 h in cell culture medium without phenol red. Excitation and emission wavelengths used for fluorescence quantification were 485 nm and 535 nm, respectively. All fluorescence measurements were corrected for background fluorescence and protein concentration. Using untreated cells as a reference, the overall anti- and pro-oxidant outcome was evaluated by comparison of five different measurements and expressed as percent of controls.

Biofilm formation ability of S. suis was monitored as described previously [26 (link)]. S. suis HA9801 was grown in TSB medium 12 h at 37°C, and then the bacterial culture was diluted with fresh TSB medium to a concentration of 10⁶ CFU/ml for the anti-biofilm assay. A PF stock solution was freshly prepared in distilled water at a concentration of 1.6 mg/ml. After filtering with a 0.22 water-based filter, the stock solution was diluted at different concentrations ranging from 6.25 to 25 μg/ml in sterile culture medium, and an equal volume was added to the above bacterial suspension and incubated at 37°C for 24 h without shaking. A control culture with S. suis and no PF was also performed. Following growth, planktonic bacteria were removed and the biofilm was stained with 1% crystal violet for 10 min and then washed with phosphate-buffered saline (PBS). After adding 95% ethanol to release the dye, the absorbance at OD595 nm was recorded with a Tecan GENios Plus microplate reader (Tecan, Austria). Biofilm formation by the ΔluxS mutant of S. suis HA9801, previously constructed by our research group [10 (link)], was assessed as described above. The assay was performed in the presence of DPD (final concentration of 3.9 μM) and PF at 25 μg/ml. A control culture with ΔluxS mutant and no PF was also performed. All assays were performed in triplicate and repeated three times.