2.11 S-mediated cell fusion assay using time-lapse microscopy and flow cytometry
For time-lapse microscopy, mRuby2 fluorescence-positive ACE2/TMPRSS2+ H1299 cells were seeded in clear bottom 24 well plates (ibidi, #82426;) overnight and then treated with DMSO or 10 µM LA for 1 h, followed by adding HEK293T cells expressing EGFP or both S protein and EGFP from a single bicistronic mRNA in (Spike-HEK293). Time-lapse fluorescence images were acquired at 4 min interval for 1 h at x40 magnification using Leica DMi8 microscope (Leica Microsystems, Germany). For flow cytometry assay, mRuby2-positivie ACE2/TMPRSS2+ H1299 cells were seeded in 12 well plate (2×105 cells/well) overnight and treated with DMSO, or 10 µM of lancemaside A, B, or D. After incubation for 30 min, 6.5×104 cells of EGFP-HEK293 or Spike-HEK293 cells were added and further incubated for 1 h at 37°C in CO2 incubator. The co-cultures were harvested using trypsin-EDTA and 1 × 104 cells and analyzed by flow cytometry using LSRFortessa (BD Biosciences) to determine the percentage of EGFP and mRuby double-positive cells as a measure of heterologous cell fusion. The data was analyzed using FlowJo software (BD Life Sciences).