Alexa fluor 594 goat anti human antibody

This assay is based on a split-GFP system (pQCXIP-GFP1–10 and pQCXIP-GFP11) where GFP signals are produced upon cell fusion (Kodaka et al., 2015 (link)). The split GFP plasmids: pQCXIP-BSR-GFP1–10 and pQCXIP-GFP11 were a gift from Yutaka Hata (Addgene plasmid #68715, https://www.addgene.org/68715/ and #68716 https://www.addgene.org/68716/). Briefly, 200,000 Vero-TMPRSS2 cells were co-transfected with 500 ng pCAGGS- spike and 500 ng pQCXIP-GFP110 or pQCXIP-GFP11 using TransIT-LT1 (Takara; MIR2300). The next day, cells were detached, pooled and reseeded in black 96-well or 24-well plates (Greiner, M0562–32EA) at 25,000 and 200,000 total cells/well, respectively. After 24 h, cells were fixed with 4% paraformaldehyde in PBS for 10 min. To measure spike expression, cells were permeabilized with 0.1% Triton X-100 for 15 min and stained with primary antibody anti-S2 (Sino Biological, 40590-D001) and secondary Alexa-Fluor 594 goat anti-human antibody (Thermo Fisher, A-11014). DAPI was used to visualize the nucleus. Images of the whole wells were obtained and analyzed using the Celigo Image Cytometer (Nexcelom). GFP signals were normalized to spike expression and DAPI counts to represent fusion activity.