Mem vitamins

Bovine aortic endothelial cells (BAECs) were isolated as previously described^{45 (link)}, and cultured in medium 199 (M-199) (Gibco) supplemented with 100 U/mL of penicillin and 100 μg/mL of streptomycin (Gibco), 1% MEM amino acids (Gibco), 1% MEM vitamins (Cellgro), 10% fetal clone III (bovine serum product, HyClone), at 37 °C with 5% CO₂. Cells at passages 5 to 8 were used for experiments. Human umbilical vein endothelial cells (HUVECs) were isolated and maintained in Medium 200 (Cascade Biologics) with low serum growth supplements. Cells at passages 2 to 4 were used. To reduce the background signals of kinases, cells were cultured for one day in serum free medium prior to the flow experiments (shear stress of 12 dyn/cm²) using a cone and plate viscometer. The cells were handled according to the regulations of the Institute of Basic Medical Sciences of the Chinese Academy of Medical Sciences, Beijing, China, and the study protocol was approved by the Institutional Review Board of the Institute of Basic Medical Sciences, the Chinese Academy of Medical Sciences. Written informed consent was obtained either from the donor or a close relative for HUVEC isolation.

All cell lines were cultured at 37 °C, 5% CO₂ in a humidified incubator according to the standard protocol of mammalian cell culturing. Human UM cells 92.1, OCM1, OMM2.3, OMM3, M20-07-070, M20-09-196, Mel290, and Mel270, authenticated by STR (Emory Genomics core facility) [22 (link)] were cultured in RPMI 1640 with L-glutamine (Corning Cellgro, Albany, NY) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO), Sodium Pyruvate (Cellgro, Albany, NY), MEM Non-Essential Amino Acids (Cellgro, Albany, NY), MEM Vitamins (Cellgro, Albany, NY), Penicillin-Streptomycin Solution (Cellgro, Albany, NY), and HEPES buffer (Corning, Albany, NY). The 92.1 and OMM2.3 cells were provided by Dr. Jerry Niederkorn (Department of Ophthalmology, UT Southwestern, Dallas, TX). The Mel290 and Mel270 cells were provided by Dr. Bruce Ksander (Schepens Eye Institute, Boston, MA). The OCM1 and OMM3 cells were donated by Dr. June Kan-Mitchel (Wayne State University, Detroit, MI). Dr. Scott Woodman (Department of Melanoma Medical Oncology and Systems Biology, MD Anderson Cancer Center, Houston, TX), Dr. Barry Burgess, and Dr. Tara McCannel (UCLA, Jules Stein Eye Institute, Calabasas, CA) isolated and provided M20-09-196 and M20-07-070 cell lines. SKOV3 ovarian cancer cells were cultured in McCoy 5 A medium with 10% fetal bovine serum and 1% penicillin and streptomycin.

Human codon-optimized SARS-CoV-2 spike-protein sequence was synthesized by MolecularCloud (MC_0101081). 5′-ACGACGGAATTCATGTTCGTCTTCCTGGTCCTG-3′ and 5′-ACGACGGAATTCTTAACAGCAGGAGCCACAGC-3′ primers were used to amplify the SARS-CoV-2 spike protein sequence without ERRS that is the last 19 amino acids^{33 (link)}. Full length and truncated SARS-CoV-2 spike-protein sequences were then cloned into pLP/VSVG plasmid from Thermo Fisher under CMV promoter after removing the VSVG sequence via EcoRI-EcoRI restriction digestion. HEK-293T cells (ATCC; mycoplasma-free low passage stock) were transfected with the expression plasmids using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol. The cells were cultured in complete RPMI 1640 medium (RPMI 1640 supplemented with 10% FBS; Atlanta Biologicals, Lawrenceville, GA), 8% GlutaMAX (Life Technologies), 8% sodium pyruvate, 8% MEM vitamins, 8% MEM nonessential amino acid, and 1% penicillin/streptomycin (all from Corning Cellgro) for 72 h^{46 (link)}. The cells were then collected using %0.05 Trypsin-0.53 mM EDTA (Corning Cellgro) and stained with Biotinylated Human ACE2/ACEH Protein, Fc,Avitag (Acro Biosystems) then stained with APC anti-human IgG Fc Antibody clone HP6017 (Biolegend). Samples were acquired on a BD FACSymphony A5 analyzer and data were analyzed using FlowJo (Tree Star).