Quantification of Cell-free DNA in Synovial Fluid

cf-DNA was measured to evaluate NET formation [28 (link)]. For quantification of cf-DNA, 200 μl of freshly extracted synovial fluid was used. Samples were incubated with 0.5 U/ml per well of micrococcal nuclease (New England Biolabs, USA) for 30 min at 37 °C. Then samples were centrifuged at 500×g for 5 min at RT. Afterwards, 100 μl of supernatant was transferred to a new well and 100 μl of Quant-iT™ PicoGreen™ dye (Thermo Fisher Scientific, MA, USA) diluted in TE buffer (10 mM Tris, 1 mM EDTA) was added. Analysis was performed using an automated Varioskan Flash Reader (Thermo Fisher Scientific, MA, USA) at 484 nm excitation and 520 nm emission, as described elsewhere [29 (link)].