WinterVomitingLab:Protocols/MNV flask infection

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Contents

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

  1. []

Video Protocol

Video protocol produced by Katie Roache:

Contact

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