WinterVomitingLab:Protocols/Propagation for MNV

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Contents

Overview

Propagating RAW 264.7 Cells for MNV-1 plaque assays using 60 mm dishes. Each T175 flask yields ~50 dishes with 80-90% confluent monolayers within 2 days.

Materials

  • RAW 264.7 cells (ATCC # TIB-71) grown to 90% confluence
  • Complete MNV DMEM
    • DMEM (Fisher- cat# SH30022LS)
    • 10% 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)
  • cell scrapers
  • beaker, stir bar and stir plate
  • 60 mm culture dishes
  • stainless steel trays for culture dishes

Procedure

  1. Calculate the number of dishes needed for the experiment and determine the amount of pre-warmed media needed (each dish will receive 5 ml of cell-containing media).
  2. Remove the flasks containing RAW cell monolayers needed to make the number of dishes required from the incubator.
  3. We use a stir plate in the cell hood and a beaker that can accommodate 800 ml of liquid at a time. This beaker (with stir bar/plate) is used for mixing the cells and fresh media before and during dish preparation.
  4. Add the appropriate amount of fresh media to the beaker.
  5. Pour media from each flask you will use into waste beaker.
  6. Add 10 ml of fresh media to each flask.
  7. Using a cell scraper, 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.
  8. Using 10 ml pipet, rinse the flask thoroughly with the media added to the flask to further remove cells from flask wall and mix the cells. A little time spent doing this will prevent cell clumping.
  9. Add the appropriate amount of cells to the beaker containing freshly aliquoted media on the stir plate for at least 5 min.
  10. While the cells are mixing, ethanol stainless steel trays, place inside hood, and fill with empty 60 mm cell culture dishes.
  11. Using 25ml pipet, aliquot 5 ml of the mixture into each dish.
  12. Label one dish on each tray with the cell line and date.
  13. Discard old flasks and place new trays of cells 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.
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  3. Anecdotal observations that might be of use to others can also be posted here.

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References

Relevant papers and books

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