WinterVomitingLab:Protocols/Propagation for MNV
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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.
- 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
- 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).
- Remove the flasks containing RAW cell monolayers needed to make the number of dishes required from the incubator.
- 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.
- Add the appropriate amount of fresh media to the beaker.
- Pour media from each flask you will use into waste beaker.
- Add 10 ml of fresh media to each flask.
- 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.
- 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.
- Add the appropriate amount of cells to the beaker containing freshly aliquoted media on the stir plate for at least 5 min.
- While the cells are mixing, ethanol stainless steel trays, place inside hood, and fill with empty 60 mm cell culture dishes.
- Using 25ml pipet, aliquot 5 ml of the mixture into each dish.
- Label one dish on each tray with the cell line and date.
- Discard old flasks and place new trays of cells in the 37° C incubator.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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