Infinite f200 pro plate reader

To quantify circulating cell-free DNA, 10 μL of plasma from r-axSpA patients and HDs was placed into 96-well black microplate and stained with 1 μM Sytox green dye (Sytox Green Nucleic Acid Stain, Invitrogen) in Tris-buffered saline [TBS (50 mM Tris-Cl, pH 7.5 and 150 mM NaCl)] at RT for 15 min in the dark. Next, plates were read in Infinite F200® Pro plate reader (Tecan Group Ltd) with a filter setting of 485 (excitation)/528 (emission). The data were analysed using a serial dilution of DNA from salmon as calibration curve.
Nucleosomes were measured by using the Human Cell Death Detection ELISAPLUS (Roche Diagnostics) according to the manufacturer’s instructions. Briefly, monoclonal antibodies against DNA (double and single strand) and histones (H1, H2A, H2B, H3 and H4) were used to detect mono- and oligo-nucleosomes in serum from r-axSpA patients and HDs. Quantification of nucleosomes was carried out by photometric determination of the absorbance at 405 nm (reference wavelength: 492 nm). Neutrophil elastase was measured by PMN Elastase Human ELISA kit (Abcam) in serum from r-axSpA patients and HDs following the manufacturer’s recommendations.

The antioxidant potential of the pineapple peel, crown and core extracts was determined based on DPPH free radical scavenging assay as previously described [14 ]. Before the analysis, 3000 µg/mL of stock sample was prepared. The assay was conducted in a 96-well microplate with 50 µL of the test samples prepared in 7 serial dilutions starting with 1000 µg/mL in the well. An aliquot of 100 µL of 0.15 mM DPPH was then added into the well. For the blank sample, 100 µL of methanol was added to obtain accurate sample absorbance. The plate was then incubated for 30 min before measuring the absorbance at 517 nm against the reagent blank by Tecan Infinite F200 Pro plate reader (Tecan Group Ltd., Männedorf, Switzerland). The analysis was performed in triplicate. The same procedure was applied for quercetin which acts as a positive control. The scavenging capacity of the test sample was calculated as the percentage of DPPH inhibition (%) = [(Ao − As)/Ao] × 100 where Ao indicated the absorbance of the reagent blank and As indicated the absorbance of the test samples. The results were expressed in IC₅₀ value (µg/mL), which denotes the concentration of sample required to scavenge 50% DPPH free radicals.