WinterVomitingLab:Protocols/MNV plaque assay

Return to WinterVomitingLab

Overview

Quantify infectivity of Murine Norovirus (MNV-1).

Materials

• 2X Complete MNV MEM
  • Mediatech Cellgro MEM (VWR- cat# 45000-586)
  • 10% Hyclone Fetal Bovine Serum (VWR- cat# 16777-006)
  • 3% HEPES (VWR- cat# 12001-710)
  • 2% Penicillin/Streptomycin (VWR- cat# 16777-164)
  • 2% Sodium Pyruvate (VWR- cat# 45000-710)
  • 2% L-Glutamine (VWR- cat# 12001-700)
  • 2% Non-essential amino acids (VWR- cat# 12001-634)
  • 3% Sodium Bicarbonate (VWR- cat# 45000-704)
• Lonza SeaKem LE Agarose (VWR- cat# 12001-870)

Procedure

1. At least 45 minutes prior to infection, place infection media, 2X MNV MEM, and a sterile 500 ml glass bottle in the 37°C water bath, and place 1% SeaKem agarose (that has been previously autoclaved and re-melted) in the 45°C water bath.
2. After making sample dilutions, aspirate growth medium from the 60 mm dishes, and add 0.4 ml of infection media to each dish.
3. Plate 100 ul of sample dilution. Place trays in 37°C incubator with 5% CO2 for 1 hour, rocking the trays every 15 minutes.
4. After 1 hour, aspirate the inoculum and media from the cells.
5. Immediately add agarose overlay as follows- mix equal amounts of 1% SeaKem agarose and 2X MNV MEM, and add 3-4 ml to each dish.
6. Allow agarose to solidify (should not take longer than 10 min).
7. Leave trays in incubator for 48-60 hr (checking for plaques at 48, 54 and 60 hr).

 * When working with a large number of 60mm dishes, we have started adding 1 ml of 1X MNV MEM on top 
     of the solidified agarose layer.  This prevents the underlying cells from drying out.

If plaques can be visualized, proceed to staining procedure with crystal violet or neutral red staining.

References

Relevant papers and books

Contact

• Who has experience with this protocol?

or instead, discuss this protocol.