WinterVomitingLab:Protocols/neutral red

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Overview

Cell staining to aid in visualizing viral plaques. Used with MNV-1, FCV and HAV plaque assays.

Materials

• Neutral Red solution (3.3 g/L) (Sigma- cat# N2889)
• Lonza SeaKem LE Agarose (VWR- cat# 12001-870)

Procedure

1. Remove liquid medium from agarose layers on virus-infected cell monolayers in 60 mm dishes (skip this step if no liquid is present).
2. Place neutral red and a sterile 500 ml glass bottle in the 37°C water bath and melted 0.5% SeaKem agarose (previously autoclaved) in the 45°C water bath for at least 45 minutes before adding 2nd overlay.
3. Mix 0.75% of the neutral red solution (see note) and 0.5% SeaKem agarose together.
4. Add 3 ml to each dish.
5. After 4-5 hours, count plaques.

 * The % of neutral red solution to add may need to be modified over time as the solution starts to
   precipitate over time.  We have used as high as 1.2% of the 3.3 g/L solution.  You start to 
   notice the color fade over time.  Storing the solution in aliquots and in the dark at 4 deg C 
   prolongs its shelf life.

References

Relevant papers and books

Contact

• Who has experience with this protocol?

or instead, discuss this protocol.