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.