WinterVomitingLab:Protocols/FCV or HAV flask infection

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Overview

Preparation of MNV-1, FCV or HAV cell culture lysate.

Materials

• Flask with a monolayer of cells grown to 80-90% confluence
  • RAW 264.7 cells for MNV-1
  • CRFK cells for FCV
  • FRhK cells for HAV
• Partially purified cell culture lysate of virus
• 1X MEM infection media
• Complete DMEM cell culture maintenance medium

Procedure

1. Place media in 37°C water bath 30-45 minutes before beginning.
2. Decant media from flask(s) into waste beaker.
3. Add 10 ml infection media (1X MEM) and ~100 ul virus stock to each flask.
4. Incubate flask(s) in 37°C incubator with 5% CO2 for 1 hour, rocking every 15 minutes.
5. After 1 hour, decant or pipette inoculum and infection media into waste beaker.
6. Add 10 ml maintenance media (DMEM) to each flask.
7. Incubate flask(s) at 37°C with 5% CO2 for 48 hours (MNV and FCV) or 1 week (HAV) or until nearly all the cells are detached.
8. After incubation, place flask(s) in -70°C freezer.
9. Perform 3 cycles of freeze/thawing of each flask to release virus from the cells.
10. Collect liquid medium and proceed to protocol for generating Partially purified virus stocks.

References

Relevant papers and books

Video Protocol

Video protocol produced by Katie Roache:

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Contact

• Who has experience with this protocol?

or instead, discuss this protocol.