2.2. Transfection and transduction of 293T cells

The 293T human embryonic kidney cells (obtained from ATCC, Manassas, VA) were maintained at 37°C under 5% CO₂ in Dulbecco's modified Eagle's medium (Wisent, St. Bruno, QC, Canada) containing 5% fetal bovine serum (VWR, Radnor, PA) and 100 μg/ml of penicillin–streptomycin (Wisent). Cells were cultured for no more than 10 passages. The plasmid expressing the full‐length SARS‐CoV‐2 Spike was kindly provided by Stefan Pöhlmann and was previously reported. 7 The 293T cells were transfected with 10 μg of Spike expressor and 2 μg of a green fluorescent protein (GFP) expressor (pIRES‐GFP) for 2 × 10⁶ 293T cells using the standard calcium phosphate method. For the generation of 293T cells stably expressing the SARS‐CoV‐2 S protein, transgenic lentiviruses were produced in 293T using a third‐generation lentiviral vector system. Briefly, 293T cells were cotransfected with two packaging plasmids (pLP1 and pLP2), an envelope plasmid (pSVCMV‐IN‐VSV‐G), and a lentiviral transfer plasmid coding for a GFP‐tagged SARS‐CoV‐2 Spike (pLV‐SARS‐CoV‐2 S C‐GFPSpark tag) (Sino Biological, Beijing, China). A supernatant containing lentiviral particles was used to transduce more 293T cells in the presence of 5 μg/ml polybrene. The 293T cells stably expressing SARS‐CoV‐2 Spike (GFP+) were sorted by flow cytometry. 10 , 11