Intracellular Flavivirus Protein Detection

Intracellular staining of viral protein was performed with True-Nuclear transcription factor buffer set (Biolegend, San Diego, USA) as described elsewhere [7 (link)]. Briefly, LHCN-M2 cells were incubated with purified pan-flavivirus 4G2 antibody (3 mg/mL–Biomanguinhos, Fiocruz, Brazil) for 1 h at 4°C followed by incubation with fluorescent secondary antibodies (Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody Alexa Fluor 546 # A-11003, or Alexa Fluor 488 # A-11001, ThermoFisher Scientific, MA, USA) for 30 min at 4°C, being then analyzed using a FACS Canto II (BD) flow cytometer, applying the FACS Diva analysis software. We also confirmed the myogenicity of the cells by the expression of CD56 by flow cytometry using an anti-CD56 PE (#555516, BD Biosciences, NJ, USA).