Flexstation equipment

The 8-OHdG levels in serum and pancreatic DNA were evaluated by ELISA using the HT 8-oxo-dG ELISA Kit II (Trevigen^®, Gaithersburg, MD, United States). Briefly, 25 μl of serum samples (1:10), DNA extracted from the pancreas (500 μg/ml), and 8-OHdG monoclonal solution were added in a pre-coated 96-well plate and incubated for 1 h at 25°C, and then washed for four times with phosphate buffered saline containing Tween 20 (PBST). Fifty μl of goat anti-mouse IgG-HRP antibody were then added (incubation for 1 h at 25°C). After four washes with PBST, 50 μl of the TACS-Sapphire^TM reagent were added in each well. After 15 min at 25°C, 50 μl of hydrochloric acid (0.2 M) were added and 8-OHdG levels were determined (450 μm) using the FlexStation^® equipment and SoftMax^® Pro software (Molecular Devices, Sunnyvale, California, United States). A standard curve (3.13 to 200 nM) for 8-OHdG was performed following the manufacturer instructions.

Endothelial cells were seeded in black-walled, clear-bottomed 96-well plates (Corning, NY, United States) at a density of 50,000 cells/well in DMEM with 10% FBS and incubated for 24 h at 37°C in a 5% CO₂. In the following day, cells were incubated with vehicle, cmDNA or dmDNA (1 μg/ml) diluted in DMEM without phenol red for 30 min. The medium was replaced for 100 μL of dye solution (Molecular Devices, Sunnyvale, CA, United States) and plates were incubated for 1 h at room temperature. Transient changes in Ca²⁺ concentration induced by adenosine 5′-triphosphate (ATP) (10^–5 M) were measured by fluorescence (515–575 nm) using the FlexStation^® equipment and SoftMax^® Pro software (Molecular Devices, Sunnyvale, CA, United States). ATP-induced responses were determined immediately upon its addition and measured as peak fluorescent intensity minus basal fluorescent intensity and the area under the curve was calculated.