Dulbecco s modified eagle medium high glucose

Human HaCaT keratinocytes (Cell Lines Service GmbH, Eppelheim, Germany) were maintained in Dulbecco's Modified Eagle Medium/high glucose (Hyclone Laboratories, South Logan, UT, USA) containing 10% fetal bovine serum (Gibco, Life Technologies Co., Grand Island, NY, USA) and an antibiotic-antimycotic (Gibco) at 5% CO₂, 37 °C, and humidified atmosphere conditions.

Plasmids expressing wild-type (WT) or mutant α2β2 or α2β4 were transiently transfected in HEK293 cells (TsA subclone; American Type Culture Collection) as reported (Conti et al., 2015 (link)). In brief, cells were cultured in DMEM high glucose (Dulbecco’s modified Eagle medium high glucose; HyClone Laboratories, Logan, UT, United States) supplemented with 10% fetal calf serum (HyClone) and 2 mM L-glutamine, at 37°C and 5% CO₂. For patch-clamp experiments, cells were seeded onto 35-mm culture dishes. Transfection was carried out with Lipofectamine 2000 (Life Technologies). To simulate the heterozygous state, equal amounts of WT and mutant plasmids were cotransfected. The DNA concentration in the transfection mixture was 1.33 ng/μL. Cells were incubated with the transfection mixture for 5 h, at 37°C, and kept at 30°C in 5% CO₂ during the 24 h preceding the electrophysiological recordings, to enhance the surface receptor density (Cooper et al., 1999 (link)).

Human embryonic kidney (HEK)293T/17 cells (ATCC: CRL11268) stably expressing a SARS-CoV-2 entry receptor of human cells, hACE2 (HEK293T/17-hACE2) were generated by lentivirus transduction as described previously [36 (link),37 (link)] and maintained in Dulbecco’s Modified Eagle Medium/high glucose (Hyclone Laboratories, Logan, UT, USA) supplemented with 10% (v/v) fetal bovine serum and antibiotics [36 (link),38 (link)].
The HIV-based lentivirus carrying a firefly luciferase reporter gene was pseudotyped with SARS-CoV-2 S protein (SARS-CoV-2 S pseudotyped HIV) by the method mentioned previously [36 (link),39 (link)]. Briefly, the infectious viral particles were generated by co-transfection of pCSFLW, pCMV-ΔR8.91, and the pCAGGS plasmid carrying codon-optimized SARS-CoV-2 S gene (GenBank: YP_009724390.1) into HEK293T/17 cells. The supernatant containing SARS-CoV-2 S pseudotyped viral particles was harvested and stored at −80 °C until used.