Varioskan equipment

HeLa-luc cells containing the luciferase gene under the control of the heat shock protein gene promoter, HSE, were plated on 24-well plates at a concentration of 15 × 10⁴ cells/ml and co-cultured with THP1 or monocytes in the indicated ratios for 20 h. Cells incubated with 5 µM U133 were used as a positive control. The luciferase reporter test was analyzed using the Bright Glo kit (Promega, Madison, WI, USA) according to the manufacturer's protocol. Luciferase activity was detected using Varioskan equipment (Thermo Fisher, Waltham, MA, USA).

An indirect ELISA protocol was initially performed (dilution chessboard) for titration of sera coming from NV, V and CN pigs using a secondary antibody Goat anti-Pig IgG (Fc): HRP (AbSerotec AAI41P) and Porcillis PRRSV vaccine as coating antigen (Intervet Lot. A200ED03) (Additional file 2). Using a range of sera dilutions previously titrated, circulating IgG antibodies from NV and CN pigs were tested in a double ELISA test against homologous NV serum-derived exosomes (sandwich ELISA) and against whole viral vaccine (Porcillis PRRS Vaccine “intervet” lot. A200ED03) as previously described. For sandwich ELISAs, plates were first coated with anti-CD63 antibodies and after washing and blocking, SEC fractions (100 uL per well) containing exosomes were incubated 90 min at 37 °C. Sera samples were afterwards incubated for 1 h at room temperature, washed and incubated with secondary antibody Goat anti-Pig IgG (Fc): HRP (AbSerotec AAI41P) at 1:10 000 dilution and optical density was measured at 450 nm using Varioskan equipment (Thermo Scientific).