Ab6749

DF-1 or A549 cells were seeded in 24-well plates (Corning) and inoculated at an MOI of 0.01 (DF-1) or 1 (A549). In case of A549 cells, spin-inoculation (800xg, 10 min) was applied [36 (link)]. After 1 hour, cells were washed once with PBS and fresh infection media was added. Subsequently, cells were incubated for 24 hours and RNA was isolated according to the manufacturer’s instructions. In addition, cells were treated with trypsin-EDTA (Lonza) and fixed in Cytofix/Cytoperm (BD, The Netherlands) for flow cytometry analysis according the manufacturer’s instructions. After fixation, cells were incubated in 1% normal goat serum (MP Biomedicals) and stained with 1:1000 anti-NDV (ab34402, Abcam, UK) and 1:1000 secondary FITC-labelled antibody (ab6749, Abcam) and analyzed by FACS Canto (BD) to determine the percentage of infected cells. qRT-PCR (45 cycles) was performed using 5 μl RNA in an ABI PRISM 7500 sequence Detection System (Life Technologies) using TaqMan gene expression assay for human IFN-β, chicken IFN-β and chicken β-actin (Hs00277188, Gg03344129, Gg03815934, Thermo Fischer Scientific). Human β-actin primers have been described before [31 (link)]. Results are shown as fold change of inoculated samples versus mock-inoculated samples both corrected for the household gene actin-β, calculated using the 2^-ΔΔ_T method [37 (link)].