Omega mars software

To quantitatively examine NF-κB activity, luciferase reporter plasmid containing five copies of an NF-κB response element (NF-κB-RE) that drives transcription of a destabilized form of NanoLuc^® luciferase (pNL3.2.NF-κB-RE, Promega Biotech AB, Nacka, Sweden) was employed. Cells were first incubated with either HC or PAH EVs for 24 h followed by reversely transfected with miR inhibitor or mimic in a 48-well plate for 24 h. Post miR transfection cells were again transfected with 50 ng pNL3.2.NF-κB-RE luciferase containing plasmid or negative control plasmid without NF-kB-RE for 24 h, before luciferase activity analysis. End-point luminescence was measured using a microplate reader (FLUOstar Omega, BMG LABTECH, Ortenberg, Germany) equipped with Omega Mars software (BMG LABTECH). Data was presented as fold change in luciferase activity as against non-EVs-exposed, control transfected cells.

Total concentrations of Nitric Oxide (NO) were measured by colorimetric assay (Thermo Fisher Scientific, Spain) following manufacturer’s instructions. Serum samples were ultrafiltered through a 10,000 molecular weight cut-off (MWCO) and diluted 1:10. Samples were quantified by reference to standard curves constructed with known amounts of Nitrate Standard solution. Absorbance was read at 540 nm using a microplate reader (LUMistar Omega, BMG Labtech) and analysed using the Omega MARS software (BMG Labtech).