Anti nemo

NEMO-reconstituted THP1 cells were treated with HMW poly(I:C) or carrier. The samples were fixed as above and stained with anti-IKKi (1:100, Cell Signaling Technology, 3416) and anti-NEMO (1:200, BD Biosciences, 611306) blocked with 5% donkey serum and incubated with donkey anti-rabbit plus and anti-mouse minus probes (Sigma-Aldrich, DUO92002, DUO92004) in situ detection reagent red (Sigma-Aldrich, DUO92008) following the manufacturer’s protocol. Images were acquired with multispectral imaging flow cytometry (40× and 60× objectives, ImageStream X Mark II flow cytometer, Amnis/EMD-Millipore), compensated, and processed using IDEAS software (Amnis). In focus, single cells were gated using area, aspect ratio, and gradient root mean squared, channel 01 (RMS Ch01). Stained cells were gated to exclude false positive signal (30 (link)); PLA⁺ specs were identified using R3 Bright Detail Intensity, corresponding to a 3-pixel signal radius. For area analysis, the 20× objective of an IncuCyte S3 (Essen) was used to acquire images, and analysis was done with IncuCyte software.