SARS-CoV-2 neutralization assay

1. Seed Vero E6 cells in a 6-well plate (approx. 4X10⁵ cells per well) and incubate overnight at 37°C;
2. Prepare samples and reagents to transport to BSL-3 suite
   1. Heat inactivate serum for 30 min at 52°C
   2. Pre-heat in 52°C water bath overlay media (DMEM - 1% NEA, 100ug/ml gentamycin)
   3. 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.
   4. Check that cells are 80-90% confluent 
   5. Prepare final overlay media (2mls/well * 6 wells/plate = 12 mls per plate)
      1. Melt 2% agarose (in water) and dilute 1:10 in preheated overlay media
   6. Transfer diluted samples, cells, dilution media, final overlay media to BSL-3 suite
3. Place final overlay media in 42°C water bath
4. Thaw prepared PRNT stocks of virus and dilute appropriately in dilution media
5. Add 115ul of diluted PRNT virus to every well in the plate of diluted samples
   1. Transfer to 37°C incubator and incubate for 1hr
6. Transfer 200ul of virus/sample mixture to 6 well plate, starting from the 1:20 dilution
   1. Change tips between samples
   2. Transfer plates to 37°C and incubate for 1hr, rocking plates every 15 min
7. Transfer 2ml of final overlay media to every well of the 6-well plate. 
   1. Incubate plates for 3 days at 37°C, 5% CO₂;
8. Fix cells by adding 2ml 10% formaldehyde in PBS and incubate at room temp for 30min;
9. Carefully remove overlay and discard in biohazardous waste;
10. Stain with 0.2% (w/v) crystal violet in 70% ethanol.

Note:

• DMEM: High glucose with L-Glu, Sodium pyruvate.