Quantification of Innate Immune Sensors

Membranes were stained as described above with additional AnnexinV-BV421 staining (Milteny Biotec). Subsequently, cells were fixed and permeabilized by a permeabilization buffer set (eBioscience) with 1% paraformaldehyde, 0.5% saponin and stained with either rabbit anti-Mx1 (ProteinTech, Chicago, USA), rabbit anti-MDA5 (Abcam, Cambridge, UK), rabbit anti-DDX58 (Abcam), rabbit anti-IFI16 (Abcam) and rabbit anti-ZBP1 (Thermofischer, Rockford, USA)) and incubated in the dark for 45 min on ice. As a secondary antibody, chicken anti-rabbit-AF488 (Invitrogen, Carlsbad, USA) was used. Unstained cells and isotype-matched controls (Becton Dickinson Biosciences) were used to assess antibody specificity. Analysis was performed as previously described [21 (link)].