<p>Gating strategy and analysis for flow cytometry of ACE2-expressing constructs bound to sarbecovirus RBDs.</p>

<p><b>(A)</b> BHK-21 cells were transfected with HA-tagged bat ACE2-expressing constructs (<a href="plosbiology.org/article/info:doi/10.1371/journal.pbio.3003944#pbio.3003944.s014" target="_blank">S2 Table</a>) and baited with His tagged-purified RBD proteins for bat sarbecoviruses. Cells were stained with anti-HA PE-conjugated antibody (ACE2) and anti-His APC-conjugated antibody (RBD). Live and singlet BHK-21 cells were gated as PE + APC+ (ACE2 + RBD+), relative to mock-transfected cells (pDISPLAY). <b>(B)</b> The SE% Dymax metric was used to calculate the percentage of ACE2 + RBD+ cells, shown for all RBDs tested with our library of bat ACE2 and human ACE2.

Representative datasets are shown for mock-transfected cells and raccoon dog ACE2 with SARS-CoV-2 RBD, with their respective SE Dymax % positive value noted. <b>(C)</b> Heatmap showing the binding of purified bat sarbecovirus proteins to ACE2s of human, <i>M. lucifugus Rh. cornutus</i> by ELISA (pink, EC₅₀ µg/ml) or flow cytometry (blue, % ACE2⁺RBD⁺ cells). <b>(D)</b> ELISA binding curves of human, <i>M. lucifugus</i> and <i>Rh. alcyone</i> ACE2s with SARS-CoV-2 and RhGB07 RBDs, depicted also as a heatmap with calculated EC₅₀ µg/ml.