CannonLab:Protocol RAW cell passage
Passaging RAW 264.7 Cells.
- RAW 264.7 cells (ATCC # TIB-71)
- Complete MNV MEM
- DMEM (Fisher- cat# SH30022LS)
- 10% HyClone Fetal Bovine Serum (VWR- cat# 16777-006)
- 1% HEPES (VWR- cat# 12001-710)
- 1% Penicillin/Streptomycin (VWR- cat# 16777-164)
- 1% Sodium Pyruvate (VWR- cat# 45000-710)
- Determine the number of new flasks to be made, and label with passage #, cell line, date, and initials. Add the appropriate amount of fresh media for the size flask you are using (T25- 7ml, T75- 25ml, T175- 50ml), minus the amount you will be adding from the passaged flask. RAW cells are passaged at a ratio of 1:5 during the week and 1:10 over weekends.
- Pour media from each flask into the waste beaker, cover beaker, and set aside.
- Add 10ml of fresh media and, using a new cell scraper for each flask, gently scrape the cells from the bottom of the flask using a side to side motion, working from the bottom of the flask to the top. DO NOT move back down in the flask once you have gone up!
- Using a pipet, rinse the flask thoroughly to remove and mix all cells, then aliquot them into the appropriate number of new flasks containing fresh media.
- Discard old flasks and place new flasks in the 37° C incubator.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
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Relevant papers and books
- Wobus CE, Karst SM, Thackray LB, Chang KO, Sosnovtsev SV, Belliot G, Krug A, Mackenzie JM, Green KY, and Virgin HW. Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages. PLoS Biol. 2004 Dec;2(12):e432. DOI:10.1371/journal.pbio.0020432 |
- Cannon JL, Papafragkou E, Park GW, Osborne J, Jaykus LA, and Vinjé J. Surrogates for the study of norovirus stability and inactivation in the environment: aA comparison of murine norovirus and feline calicivirus. J Food Prot. 2006 Nov;69(11):2761-5.
- Gonzalez-Hernandez MB, Bragazzi Cunha J, and Wobus CE. Plaque assay for murine norovirus. J Vis Exp. 2012 Aug 22(66):e4297. DOI:10.3791/4297 |
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