Recombinant ifn a d

4 × 10⁴ cells per well were seeded on a 24‐well plate. One day later, 200 ng of Cy5‐labelled dsRNA‐RL or Cy5‐dsRNA‐GFP or 15 pmol siRNA‐GFP (Silencer GFP siRNA, Ambion) or 15 pmol siRNA control (Silencer negative control siRNA # 1, Ambion) or transfection reagent alone (mock) was transfected into cells using Lipofectamine 2000 (Invitrogen). To test the effect of IFN on dsRNA/siRNA‐mediated gene silencing, 200 U of recombinant IFN A/D (PBL Assay Science) was added at the time of seeding the cells. To block the IFN receptor, an anti‐IFNAR antibody (LEAF purified anti‐mouse IFNAR‐1 antibody, Biolegend) was included at 10 μg ml⁻¹. An isotype‐matched irrelevant specificity antibody (LEAF purified mouse IgG1, k isotype control antibody, Biolegend) was used as a control. For analysis, cells were recovered by trypsinisation, washed in PBS and resuspended in FACS buffer (PBS, 1% FCS, 5 mM EDTA) containing the live/dead cell discriminator dye DAPI, as well as a fixed amount of fluorescent reference beads (Coulter CC Size standard L10, Beckman Coulter) in order to allow for flow cytometry‐based cell counting. Flow cytometric analyses were performed with an LSRFortessa (BD Biosciences) acquiring a sample size of 10,000 cells to meet statistical robustness. Data were analysed using FlowJo (Tree Star).