Tristar multimode microplate reader lb941

To investigate the effect of FH on the interaction between neutrophils and endothelial cells, adhesion assays were performed. Endothelial monolayers were grown on wells of Costar 96-well black transparent-bottom plates (Corning Life Sciences, Corning, NY, USA) and pretreated with 50 µg/ml FH, human serum albumin (HSA), or medium for 1 h at 37°C. Prepared neutrophils were suspended in RPMI 1640 medium without serum to a concentration of 1 × 10⁶ cells/ml and then stained with 5 µM Cell tracker green (Invitrogen) for 45 min at 37°C. After washing, neutrophils in serum-free ECM were primed with 2 ng/ml TNF-α and then added to the wells to adhere for 1 h at 37°C. In some experiments, TNF-α primed neutrophils were pre-incubated with 100 µg/ml FH or controls for 1 h at 37°C, followed by added to non-treated hGEnC monolayers. Non-adherent cells were removed by extensive washing. Adherent cells were reflected by fluorescence intensity (FI) of neutrophils measured by a fluorescence reader (TriStar Multimode Microplate Reader LB941, Berthold Technologies, Bad Wildbad, Germany) with filters of 495 nm (excitation) and 515 nm (emission).

Reporter gene assays were routinely performed in our laboratory using pcDNA-ΔLf or pcDNA-hLf constructs or a null vector and HEK 293 cells [15] (link), [41] (link), [42] . The reporter pGL3-SelH-Luc vector was obtained as in [13] (link) except that the 167 bp SelH promoter fragment was amplified with the primer pair listed in Table S1, cloned into the pGL3-promoter-Luc vector (Promega) and sequenced before use. HEK 293 cells were transfected (250 ng of DNA for 2×10⁵ cells, 50 ng of reporter vector and 200 ng of ΔLf, hLf expression vector or null vector) using DreamFect (OZ Biosciences, Marseille, France). Cell lysates were assayed using a luciferase assay kit (Promega) in a Tristar multimode microplate reader LB 941 (Berthold Technologies, Bad Wildbab, Germany). Relative luciferase activities were normalized to basal luciferase expression as described [13] (link) and expressed as fold increase to the relative luciferase activity of ΔLf or hLf. Basal luciferase expression was assayed using a null vector and was determined for each vector. Each experiment represents at least three sets of independent triplicates.

NETs formation was quantified using PicoGreen as described previously [27 (link)]. Lambda DNA of known concentration was serially diluted with Tris-EDTA (TE) buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) to create standard DNA samples. To follow NETs formation, 100 μl fresh neutrophils (1 × 10⁵cells) were seeded in Costar 96-well black plates (Corning, Tewksbury, MA, USA) in the presence of 0.5 % FBS. Neutrophils were pretreated with buffer or 10 ng/ml HMGB1 for 30 min followed by stimulation with MPO-ANCA-positive IgG, or PR3-ANCA-positive IgG, or normal IgGs in an incubator containing 5 % CO₂ at 37 °C for 3 h, respectively. For assay of the role of candidate receptors, certain groups of neutrophils were preincubated with relevant reagents for 30 min on ice before being pretreated with HMGB1. Some 100 μl of the PicoGreen dye diluted 1:200 in TE buffer was added to the microplate in order to make a final volume of 200 μl per well. Following incubation in the dark for 5 min at room temperature, the fluorescent signal of the sample was measured using the microplate fluorescence reader (TriStar Multimode Microplate Reader LB941, Berthold Technologies, Bad Wildbad, Germany), at an excitation wavelength of 480 nm and an emission wavelength of 530 nm.