WinterVomitingLab:Protocols/Propagation for FCV or HAV

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Overview

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.

Materials

• CrFK (ATCC # CCL-94)or FRhK-4 (ATCC # CRL-1688) cells grown to 90% confluence
• Complete FCV/HAV MEM
  • DMEM (Fisher- cat# SH30022LS)
  • 10% Atlanta Bio Fetal Bovine Serum (Atlanta Biologicals- cat# S11050H)
  • 1% Penicillin/Streptomycin (VWR- cat# 12001-692)
  • 1% L-Glutamine (VWR- cat# 16777-162)
  • 1% Non-essential amino acids (VWR- cat# 12001-634)
• PBS
• Trypsin
• 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 a waste beaker.
6. Rinse flasks with 10 ml of PBS, pouring rinsate into waste beaker.
7. Add 3 ml trypsin (T175 flasks) and rock flask to cover the bottom with a thin layer of trypsin.
8. 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.
9. 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.
10. Add the appropriate amount of cells to the beaker containing freshly aliquoted media on the stir plate for at least 5 min.
11. While the cells are mixing, ethanol stainless steel trays, place inside hood, and fill with empty 60 mm cell culture dishes.
12. Using 25ml pipet, aliquot 5 ml of the mixture into each dish.
13. Label one dish on each tray with the cell line and date.
14. After all trays have been made, place trays in the floor incubator in the main lab.
15. Discard old flasks and place new trays of cells in the 37° C incubator.

Notes

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References

Relevant papers and books

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