CannonLab:Protocol RAW cell passage

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Contents

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

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

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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. . pmid:15562321. PubMed HubMed [Paper1]
  2. Cannon JL, Papafragkou E, Park GW, Osborne J, Jaykus LA, and Vinjé J. . pmid:17133824. PubMed HubMed [Paper2]
  3. Gonzalez-Hernandez MB, Bragazzi Cunha J, and Wobus CE. . pmid:22951568. PubMed HubMed [Paper3]
All Medline abstracts: PubMed HubMed

Contact

  • Who has experience with this protocol?

or instead, discuss this protocol.


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