Sunrise rc st evolyzer plate reader

As previously described, serum NETs levels were assessed using a neutrophil elastase enzyme-linked immunosorbent assay (ELISA) (17 (link)) at baseline and 3 months after recruitment. Briefly, mouse anti-human neutrophil elastase (Calbiochem, Darmstadt, Germany) was diluted 1:2000 in coating buffer from the cell death detection ELISA kit (Roche, Basil, Switzerland) and incubated in high-binding 96-well plates overnight at 4 °C to generate mouse anti-human neutrophil elastase-coated plates. Plates were washed three times with PBS/Tween20 and then blocked with 1% bovine serum albumin (BSA) in PBS for 6 h at room temperature. Afterward, serum samples (1:10 in 1% BSA) were added to the plates and incubated overnight at 4 °C. Following three washes with PBS/Tween20 solution, the samples were incubated with anti-human DNA-peroxidase antibody (1:10 dilution in incubation buffer) from the cell death detection ELISA kit (Roche) for 1 h at room temperature. Next, the plates were washed five times with PBS/Tween20 before adding TMB substrate (Thermofisher Scientific, Waltham, MA, USA), followed by a stop solution. The plates were read at 450 nm, and the optic density index was calculated as previously described (17 (link)) in a Sunrise-RC/ST Evolyzer Plate Reader (Tecan Life Sciences, Männendorf, Switzerland).