WinterVomitingLab:Protocols/Passaging A549 cells
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Passaging A549 Cells.
- A549 (Crandell Feline Kidney) cells (ATCC # CCL-185)
- Complete A549 F-12K Medium
- F-12K (Fisher- cat# MT10025CV)
- 10% Bovine Growth Serum (VWR- cat# 82013-586)
- 1% Penicillin/Streptomycin (VWR- cat# 12001-692)
- 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. A549 cells are passaged at a ratio of 1:5.
- Pour media from each flask into the waste beaker, cover beaker, and set aside.
- Rinse flasks with PBS, collecting the rinsate in the waste beaker.
- Add 3 ml trypsin (T175 flasks) and rock flask to cover the bottom with a thin layer of trypsin.
- Place flasks in 37°C incubator, checking every 2 minutes, until all cells have detached from the bottom. If the cells do not detach easily, gentle agitation may be necessary.
- Add 7 ml of fresh media and thoroughly rinse the flask to remove and mix all cells, then aliquot them into the appropriate number of new flasks containing fresh media.
- Discard old flasks and place new flasks 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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