Opera phenix imaging platform

Live imaging of cell viability during the course of TNF treatment was performed by incubating the cultures with NUCLEAR-ID Blue/Red cell viability reagent (Enzo) diluted in culture media for 30 min, according to the manufacturer’s instructions. Cultures were washed in fresh media before images were acquired on an Opera Phenix imaging platform (Perkin Elmer) at 37°C and 5% CO₂. For each timepoint, 37 fields of view were imaged at 20× objective in technical duplicate cultures of each treatment condition, with DAPI (ex375/em435–480) and dsRed (ex561/em570–630) laser and filter sets. For image processing, the number of red nuclei (indicating dead cells) was divided by the number of blue nuclei (indicating total cells) within each field of view to obtain a measure of the proportion of dead nuclei per condition.

HAP1 control or HAP1 USP18 KO cells were seeded in fluorescence compatible 96-well plates (Corning), treated with IFN for the indicated times, subsequently washed with PBS, fixed using 4% paraformaldehyde (sc-281692, ChemCruz) for 10 min, permeabilised using 100% methanol for 10 min at −20 °C, and blocked with 5% BSA in PBS for one hour at room temperature. The primary antibodies were diluted in PBST (PBS + 0.05% Tween20) to the following concentrations: USP18 (1:100), ISG15 (1:100) and pSTAT1 (1:400), added and incubated overnight at 4 °C. Cells were then washed with PBST and subsequently incubated with a secondary antibody mix consisting of Goat anti-rabbit conjugated alexa-647 (1:10,000) and 4’,6’-diamidino-2-phenylindole (Dapi) (2.5 μg/mL), for 1 h at room temperature. Plates were imaged using the Opera Phenix imaging platform (Perkin Elmer).
For dsRNA analysis, the same workflow was used with the following modification: The cells were fixed and permeabilised using Cytofix/Cytoperm buffers (BD biosciences), acording to the manufacturer’s instructions. dsRNA antibody was used at a 1:100 dilution and the secondary donkey anti-mouse at a 1:500 dilution.