WinterVomitingLab:Protocols/FCV or HAV flask infection
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Preparation of MNV-1, FCV or HAV cell culture lysate.
- 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
- Place media in 37°C water bath 30-45 minutes before beginning.
- Decant media from flask(s) into waste beaker.
- Add 10 ml infection media (1X MEM) and ~100 ul virus stock to each flask.
- Incubate flask(s) in 37°C incubator with 5% CO2 for 1 hour, rocking every 15 minutes.
- After 1 hour, decant or pipette inoculum and infection media into waste beaker.
- Add 10 ml maintenance media (DMEM) to each flask.
- 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.
- After incubation, place flask(s) in -70°C freezer.
- Perform 3 cycles of freeze/thawing of each flask to release virus from the cells.
- Collect liquid medium and proceed to protocol for generating Partially purified virus stocks.
Relevant papers and books
Video protocol produced by Katie Roache:
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