Pe labeled goat anti mouse igg fc antibody

To examine hACE2 expression, 3T3 cells were transduced by AAV6/hACE2 or AAV9/hACE2 for 48 hours and dissociated by Versene solution (Thermo Fisher Scientific, MA, USA) at 37°C for 30 minutes. For each test, 2×10⁵ cells were resuspended in 100 μl of Dulbecco’s phosphate buffered saline (DPBS) containing 1% FBS and then incubated with 0.2 μg of recombinant SARS-CoV-2 Spike protein (Sino Biological, Beijing, China) for 1 hour on ice. The recombinant protein consists of the receptor binding domain (RBD) of SARS-CoV-2 Spike protein and a mouse Fc region at the C-terminus. Next, the cells were incubated with 100 μl PE-labeled goat anti-mouse IgG-Fc antibody (Jackson Immuno Research, PA, USA) for 30 minutes. After the staining procedure, cells were resuspended again in 300 μl of 1% FBS-DPBS containing 0.25 μg of 7-amino-actinomycin D (Thermo Fisher Scientific, CA, USA) to exclude non-viable cells, and then analyzed by LSRII flow cytometer (BD Biosciences, MA, USA).