Infinite m200 microplate analyzer

For the determination of the extracellular ATP, a luciferin–luciferase assay (ENLITEN ATP Assay System Bioluminescence Detection Kit, Promega) and a TECAN infinite M200 Microplate Analyzer (Lausanne, Switzerland) were used to record relative light units (RLU) as described by Liu, Sabirov, and Okada (2008) (link). As previously described (Xu et al., 2014 ), the assay was performed during 60 min in DMEM without serum or pH indicator at 37℃. The ecto-ATPase inhibitor ARL67156 was present in all experiments. Then, the medium was rapidly cooled to room temperature for the ATP assay and mixed with a 100 -μl aliquot of luciferin–luciferase reagent dissolved in dilution buffer provided with the assay kit, and chemiluminescence values (RLU) were recorded as an indication of ATP content. Even in the absence of any known ATP, considerable relative high RLU readings were caused by the medium or tissue. This appeared larger than the previously measured 1,200–1,600 RLU under control conditions (Xu et al., 2014 ), perhaps reflecting some effects of the reagents other than siRNA itself with which the cells had been treated.

A total of 236 pig serum samples were also collected from 7 farms in Lagos State in May–June 2014. Blood samples (5–10 mL) from pigs were drawn from the femoral artery after palpation of the pulse by pulling the rare leg in dorsal recumbency or at slaughter. Blood samples were stored in Giostyle sample coolers (Gio’Style, Urgnano, Italy) stocked with ice packs before being transported to the laboratory. After coagulation at room temperature, serum was separated via centrifugation at 4000 rpm for 10 min and stored at −80 °C.
The presence of antibodies raised against the nucleocapsid protein (NP) of influenza A virus was tested via competition ELISA (AI MultiS-Screen Ab Test; Idexx, Hoofddorp, The Netherlands) following the manufacturer’s instructions. The assay was carried out using both test kit controls and in-house test controls with serum of pigs experimentally inoculated with A/swine/Ontario/33853/05 (H3N2; positive control) and field swine sera from Luxembourg that tested negative via virus neutralization against a panel of representative strains (negative control). Optical density (OD) values were measured with Infinite M200 microplate analyzer (Tecan, Mechelen, Belgium).