WinterVomitingLab:Protocols/RAW 264.7 cells

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Contents

Overview

Passaging RAW 264.7 Cells.

Materials

  • RAW 264.7 cells (ATCC # TIB-71)
  • Complete MNV DMEM
    • DMEM (Fisher- cat# SH30022LS)
    • 10% HyClone FetalClone III Serum (VWR- cat# 16777-240)
    • 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

Error fetching PMID 15562321:
Error fetching PMID 17133824:
Error fetching PMID 22951568:
  1. Error fetching PMID 15562321: [Paper1]
  2. Error fetching PMID 17133824: [Paper2]
  3. Error fetching PMID 22951568: [Paper3]
All Medline abstracts: PubMed HubMed


Video Protocol

Video protocol produced by Katie Roache:

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