| Subculturing |
Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
-
Remove and discard culture medium.
-
Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum, which contains trypsin inhibitor.
-
Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 10 minutes). Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C.to facilitate dispersal.
- Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
-
Add appropriate aliquots of the cell suspension to new culture vessels.
-
Incubate cultures at 37°C.
Subcultivation Ratio: A subcultivation ratio of 1:3 to 1:8 is recommended
Medium Renewal: 2 to 3 times per week |
| References |
Crandell RA, et al. Development, characterization, and viral susceptibility of a feline (Felis catus) renal cell line (CRFK). In Vitro 9: 176-185, 1973. PubMed: 4130570
Fabricant CG, Rich LJ. Microbial studies of feline urolithiasis. J. Am. Vet. Med. Assoc. 158: 976-980, 1971. PubMed: 4930310
Lee KM, et al. Use of an established cat cell line for investigation and quantitation of feline oncornaviruses. J. Natl. Cancer Inst. 49: 55-60, 1972. PubMed: 4338781
Loffler S, et al. CD9, a tetraspan transmembrane protein, renders cells susceptible to canine distemper virus. J. Virol. 71: 42-49, 1997. PubMed: 8985321
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