WinterVomitingLab:Protocols/Propagation for FCV or HAV
Return to WinterVomitingLab
Propagating CRFK or FRhK-4 cells for plaque assays with FCV or HAV using 60 mm dishes. Each T175 flask yields ~30 FCV dishes or ~20 HAV dishes with 80-90% confluent monolayers within 2 days. CRFK cells are used for FCV infection and FRhK-4 cells are used for HAV infection.
- CRFK (ATCC # CCL-94)or FRhK-4 (ATCC # CRL-1688) cells grown to 90% confluence
- Complete FCV/HAV MEM
- DMEM (Fisher- cat# SH30081LS)
- 10% Atlanta Bio Fetal Bovine Serum (Atlanta Biologicals- cat# S11050H)
- 1% Penicillin/Streptomycin (VWR- cat# 12001-692)
- 1% L-Glutamine (VWR- cat# 12001-700)
- 1% Non-essential amino acids (VWR- cat# 12001-634)
- 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 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 a waste beaker.
- Rinse flasks with 10 ml of PBS, pouring rinsate into 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 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.
- After all trays have been made, place trays in the floor incubator in the main lab.
- 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!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
Relevant papers and books
- Who has experience with this protocol?
or instead, discuss this protocol.