175 cm2 culture flask

Human adipose tissue-derived mesenchymal stem cells (AdMSCs) and human umbilical vein endothelial cells (HUVECs) were purchased from CEFO (www.cefobio.com), Seoul, Republic of Korea. Human dermal fibroblasts were obtained from Invitrogen (Carlsbad, CA, USA). The cells were cultivated in culture medium (DMEM-low glucose containing 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin) in a 75-cm² culture flask (Nunc, Roskilde, Denmark) at 37 °C in a 5% CO₂-containing atmosphere. They were subcultured in a 175-cm² culture flask (Nunc) for expansion after they had reached approximately 90% confluence. The medium was changed every three or four days. For selenium treatment, AdMSCs were cultured with 5 ng/mL sodium selenite (Sigma Chemical Co., St. Louis, MO, USA). The cells obtained from 3 donors were used for the present study. For the inflammatory response, THP-1 cells, a human monocytic cell line derived from an acute monocytic leukemia patient were used. The THP-1 cells were cultured and maintained in RPMI medium supplemented with 10% FBS and 1% P/S at 37 °C in a 5% CO₂ environment (all from Invitrogen). To stimulate an inflammatory response, the THP-1 cells were treated with 100 ng/mL LPS.

Pseudotyped retroviruses expressing a luciferase reporter gene were prepared by co-transfecting HEK293T cells with a plasmid encoding an Env-defective and luciferace-encoding HIV-1 genome (pNL4-3.luc.R-E-) and a plasmid encoding either SARS-CoV-2 Spike, SARS-CoV-1 Spike, WIV1-CoV Spike, MERS-CoV Spike, or vesicular stomatitis virus glycoprotein (VSV-G). Transfections were performed with 15 µg of each plasmid in a 175 cm² culture flask (Nunc) using Lipofectamine 3000 (Thermo Fisher). Medium-containing transfection complexes was replaced with fresh complete medium after 8 h. Secreted pseudovirus particles were harvested from HEK293T supernatant 72 h after transfection. Pseudovirus stocks were stored at −80 °C in single-use aliquots and were titered using the Reed-Muench method to determine TCID₅₀ concentrations^{31 (link)}.