Seed Vero E6 cells in a 6-well plate (approx. 4X105 cells per well) and incubate overnight at 37°C;
Prepare samples and reagents to transport to BSL-3 suite
Heat inactivate serum for 30 min at 52°C
Pre-heat in 52°C water bath overlay media (DMEM - 1% NEA, 100ug/ml gentamycin)
Serially dilute serum in DMEM (1% FBS) in 96-well plate beginning with 23ul serum à 207ul dilution media. 2-fold dilutions are prepared by transferring 115ul into 115ul of dilution media. Be sure to remove the final 115ul from the last dilution.
Check that cells are 80-90% confluent
Prepare final overlay media (2mls/well * 6 wells/plate = 12 mls per plate)
Melt 2% agarose (in water) and dilute 1:10 in preheated overlay media
Transfer diluted samples, cells, dilution media, final overlay media to BSL-3 suite
Place final overlay media in 42°C water bath
Thaw prepared PRNT stocks of virus and dilute appropriately in dilution media
Add 115ul of diluted PRNT virus to every well in the plate of diluted samples
Transfer to 37°C incubator and incubate for 1hr
Transfer 200ul of virus/sample mixture to 6 well plate, starting from the 1:20 dilution
Change tips between samples
Transfer plates to 37°C and incubate for 1hr, rocking plates every 15 min
Transfer 2ml of final overlay media to every well of the 6-well plate.
Incubate plates for 3 days at 37°C, 5% CO2;
Fix cells by adding 2ml 10% formaldehyde in PBS and incubate at room temp for 30min;
Carefully remove overlay and discard in biohazardous waste;
Stain with 0.2% (w/v) crystal violet in 70% ethanol.
Note:
DMEM: High glucose with L-Glu, Sodium pyruvate.
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How to cite:
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
Erasmus, J. H., Khhar, A. P., O’Connor, M. A., Walls, A. C., Hemann, E. A., Murapa, P., Archer, J., Leventhal, S., Fuller, J. T., Lewis, T. B., Draves, K. E., Rall, S., Guerriero, K. A., Duthie, M. S., Carter, D., Reed, S. G., Hawman, D. W., Feldmann, H., Jr., M. G., Veesler, D., Berglund, P. and Fuller, D. H.(2020). An Alphavirus-derived replicon RNA vaccine induces SARS-CoV-2 neutralizing antibody and T cell responses in mice and nonhuman primates . Science Translational Medicine 12(555). DOI: 10.1126/scitranslmed.abc9396
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