Quantifying EV-A71 Infection and AIM2 Expression

EV-A71/13903-infected SK-N-SH and AIM2-knockdown SK-N-SH cells (SK-N-SH/siAIM2) cells at MOI of 1, and mock-infected cells were triple-stained for EV-A71 antigens, AIM2 and 7 amino-actinomycin (7-AAD). Previously shown to be useful as a biomarker for pyroptosis, 7-AAD is able to enter nuclear membranes to stain DNA following the loss of membrane integrity^{41 (link), 65 (link), 91 (link)}. Briefly, infected and mock-infected cells were collected in 1.5 ml microcentrifuge tubes and incubated for 5 minutes with 7-AAD^{87 (link)–89 (link)}. The cell pellets were then fixed with Fluorofix buffer and stained for viral antigen and AIM2 protein were using mouse monoclonal Enterovirus blend 3321 antibody and rabbit polyclonal AIM2 K-12 antibody (dilution 1:500; Santa Cruz, USA), followed by incubation with by goat-anti mouse IgG conjugated with Alexa-fluor 488 and goat anti-rabbit IgG conjugated with Alexa-fluor 546 (Molecular Probes, USA) for 30 min at RT in the dark^{92 (link)}. Flow cytometry analysis to assess localization of viral antigens and AIM2 or 7-AAD was performed as before.