2.12 Split-GFP assay to detect the S-mediated syncytia formation
ACE2/TMPRSS2+ H1299 cells separately expressing two non-fluorescent fragments of GFP (GFP1-10 and GFP11)(Buchrieser et al., 2021) were established and equal numbers of these cells were plated in clear bottom 96 well plates (ibidi, #89626). Next day, the cultures were infected with lentiviral particles expressing SARS-CoV-2 S protein for 6 h and treated with DMSO or varying concentration of LA, followed by incubation for 36 h. The cells were then fixed with 4% paraformaldehyde (PFA) and stained with DAPI (Invitrogen, D1306) to identify the nuclei. Acquisition and automated analysis of the fluorescent cell images were carried out using an ImageXpress Pico system (Molecular Devices) with CellReporterXpress software (cell scoring function, 2 channel assay for scoring cells based on the DAPI stained nuclei and GFP images). Nuclei which overlap with GFP fluorescence (designated as syncytia) and free nuclei were pseudocolored in green and red respectively using CellReporterXpress software and the percentage of syncytia formation was calculated as the ratio of green to (green + red) colored nuclei.