CannonLab:Protocol RAW cell passage

Overview

Passaging RAW 264.7 Cells.

Materials

• 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)

Procedure

1. 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.
2. Pour media from each flask into the waste beaker, cover beaker, and set aside.
3. 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!
4. 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.
5. Discard old flasks and place new flasks in the 37° C incubator.

Notes

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References

Relevant papers and books

1. 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 |
2. 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. DOI:10.4315/0362-028x-69.11.2761 |
3. 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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