Sphingosine

Human nasal epithelial cells were obtained from healthy volunteers by nasal brushings with a small brush. The cells were suspended immediately in HEPES/saline (H/S; 132 mm NaCl, 20 mm HEPES, pH 7.4, 5 mm KCl, 1 mm CaCl₂, 0.7 mm MgCl₂, 0.8 mm MgSO₄), washed once, and resuspended in H/S. The cells were treated with 0.25, 0.5, 1, 2, or 5 μm sphingosine (Avanti Polar Lipids, catalog no. 860490) or left untreated for 30 min, pelleted, and resuspended in minimum essential medium (MEM) supplemented with 10% FCS containing pp-VSV–SARS–CoV-2 spike and the same concentration of sphingosine as used in the preincubation period. The cells were then infected for 60 min, washed once in H/S, and cultured for 24 h in minimum Eagle's medium supplemented with 10% FCS to allow expression of the eGFP encoded by the particles. Infection was analyzed on a Leica TCS-SP5 confocal microscope by counting the percentage of eGFP-positive epithelial cells in at least 500 epithelial cells per sample in randomly chosen microscopic fields.
The local ethics committee of the University Hospital Essen approved the experiments under permission number 20-9348-BO. The studies were performed in accordance with the Declaration of Helsinki principles.