Pcdna3.1 hace2 plasmid

HEK293T cells (American Type Culture Collection, Manassas, VA, USA) were maintained in high glucose Dulbecco’s Modified Eagle Medium (DMEM) containing 1% penicillin/streptomycin and 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA; herein called culture medium) at 37 °C in an incubator under a water-saturated atmosphere and 5% CO₂. The culture medium was freshly provided every other day. HEK293T cells overexpressing human ACE2 (hACE2) were prepared by transfection with the pcDNA3.1-hACE2 plasmid (Addgene, Watertown, MA, USA). The cell viability was detected following methodology as described previously [23 (link)], except the absorbance here was measured at 490 nm.

HEK293T cells were seeded at 1.7 × 10⁵ cells/cm² within 10 mL of DMEM with 10% FBS and 1% penicillin-streptomycin in a T-75 flask. The next day, 5 µL of pcDNA3.1-hACE2 plasmid (Addgene) was mixed with 0.1 mL of PEI for 30 min and then added to the cells for transfection. The culture medium was replaced with a fresh one. After 48 h, 500 µg/mL G418 disulfate (Sigma-Aldrich) was used for selection for up to four weeks. The stable HEK293T cell line expressing hACE2 (HEK293T-ACE2) on the surface of the outer cell membrane was confirmed by incubating HEK293T-ACE2 cells with anti-ACE2 mAb for 1 h at room temperature. Following three gentle PBS washes, cells were treated for 25 min at room temperature with a fluorescent secondary antibody: anti-mouse IgGκ binding protein conjugated with red fluorescent dye CruzFluor^TM 594 (Santa Cruz Biotechnology). A Leica DMi8 microscope equipped with a Leica EC3 camera (Leica Microsystems, Wetzlar, Germany) was used to image the cells. Western blot analysis was performed in HEK293T-ACE2 cells by using RIPA lysis buffer (Thermo Fisher Scientific). Briefly, after performing SDS-PAGE for the cell lysate, the gel was transferred onto the nitrocellulose membrane. ACE2 mAb and HRP-conjugated mouse IgG secondary antibodies were used for Western blot detection.