Nters for Illness Control and Prevention and BEI Resources. To generate the passage 1 (P1) virus stock, Vero E6 cells, pre-seeded the day ahead of at a density of 10 million cells, have been infected in T175 flasks together with the master stock, diluted in 10 mL final volume of Opti-MEM. Following virus adsorption for the cells at 37 for 1 hr, 15 mL DMEM containing 10 FBS and 1x penicillin/streptomycin was added for the flask. The next day, media was removed, cell had been rinsed with 1x PBS and 25 mL of fresh DMEM containing 2 FBS was added. Two days later, when the cytopathic effect of the virus was clearly visible, culture medium was collected, filtered via a 0.2 mm filter, and stored at 0C. Our P2 functioning stock from the virus was ready by infecting Vero E6 cells together with the P1 stock, at a multiplicity of infection (MOI) of 0.1. Cell culture media was harvested at day two and day 3 post infection, and after the last harvest, ultracentrifuged (Beckman Coulter Optima L-100k; SW32 Ti rotor) for two hr at 25,000 rpm more than a 20 sucrose cushion. Following centrifugation, the media and sucrose have been discarded and pellets were left to dry for 5 min at space temperature. Pellets have been then resuspended over evening at 4 in 500 mL of 1x PBS. The subsequent day, concentrated virions have been aliquoted at stored at 0 . The titer of our viral stock was determined by plaque assay. Vero E6 cells had been seeded into a 12well plate at a density of two.five 105 cells per effectively, and infected the following day with serial 10-fold dilutions from the virus stock for 1 hr at 37 . Following virus adsorption, 1 mL of CXCR4 Gene ID overlay media, consisting of 2x DMEM supplemented with 4 FBS and mixed at a 1:1 ratio with 1.2 Avicel (DuPont; RC581), was added in every single well. Three days later, the overlay medium was removed, the cell monolayer was washed with 1x PBS and fixed for 30 min at space temperature with four paraformaldehyde. Fixed cells had been then washed with 1x PBS and stained for 1 hr at space temperature with 0.1 crystal violet ready in 10 ethanol/water. After rinsing with tap water, the amount of plaques had been counted as well as the virus titer was calculated. The titer of our P2 virus stock was four 108 PFU/mL.SARS-CoV-2 cholesterol depletion assayThe day prior to infection, A549 expressing hACE2 and hTMPRSS2 cells had been seeded at a density of two 104 per properly inside a poly-L-lysine coated flat-bottom 96-well plate. The following day, MBCD initial stock was prepared at a concentration of 20 mM in 1x PBS and 2-fold serial dilutions were then produced using 1x PBS. Prior to infecting cells, a 1 hr pretreatment of MBCD with SARS-CoV-2 virus or cells was carried out. Viral pretreatment was performed by mixing 25 mL of each and every MBCD dilution with 25 mL of SARS-CoV-2 (MOI of 0.5; 1 104 PFU) per effectively then incubated for 1 hr at 37 . For cell pretreatment, media was Aryl Hydrocarbon Receptor web removed from wells and cells were washed as soon as with 1x PBS. Cells have been then incubated for 1 hr at 37 with 25 mL of every MBCD dilution additional diluted in 25 mL of 1x PBS.Sanders, Jumper, Ackerman, et al. eLife 2021;10:e65962. DOI: https://doi.org/10.7554/eLife.34 ofResearch articleCell BiologyFollowing pretreatments, media or PBS/MBCD mixes have been removed from wells and wells have been washed twice with 1x PBS. Untreated and MBCD-treated cells were then infected for 1 hr at 37 with 50 mL of SARS-CoV-2 pretreated with MBCD, or with untreated SARS-CoV-2 (MOI of 0.five), respectively. One hour following virus adsorption, media was removed, cells have been washed twice with PBS, and 200 mL of DMEM containi.
