Goat anti human ig apc

HEK293T cell lines expressing hACE2 were cultured as previously described, in the presence of 3 µg ml⁻¹ puromycin to select for hACE2 expression [15 (link)]. hACE2 expression was detected using mouse monoclonal antibody against c-Myc-FITC antibody (clone 9E10) from Thermo-Fisher. Original HEK293T cell lines were used as a control. Binding affinity of RBD-Fc [11 (link)] variants were detected by staining HEK293T-hACE2 cells as previously described [15 (link)] (electronic supplementary material, figure S3). Briefly, 10⁶ cells were incubated (15 min, room temperature) with 0.5 µg ml⁻¹ of RBD-Fc variants in PBS containing 0.5% bovine serum albumin (BSA) (Thermo-Fisher). The cells were washed with PBS, 0.5% BSA and RBD-Fc binding was detected with goat anti-human-Ig-APC (Jackson ImmunoResearch Labs, Pennsylvania, USA). Data were acquired using flow cytometry using an LSRII (BD Biosciences, California, USA) and analysed using the FlowJo software (FlowJo, LLC).

HEK293T cells were transfected with plasmids encoding ACE2 orthologs or human DPP4 using PEI 40K (Polysciences) according to manufacturer’s instructions. 48h post transfection, transfected cells were detached and incubated with 5 μg/ml RBD-Fc, and the interaction was detected with goat-anti-human-Ig-APC (Jackson ImmunoResearch Laboratories, Inc). Expression of ACE2 orthologs were detected using mouse monoclonal antibody against c-Myc antibody 9E10 (Thermo Fisher) and Goat-anti-mouse FITC (Jackson ImmunoResearch Laboratories, Inc). Samples were analyzed by flow cytometry (BD Accuri C6 Flow Cytometry) and data was analyzed using FlowJo (FlowJo, LLC).