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Volumes used in this protocol are for a 75 cm² flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. It is important to subculture the cells as soon as they become confluent, or else the cells will begin to die.

1. Remove and discard culture medium. 
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53mM EDTA solution to remove all traces of serum, which contains trypsin inhibitor.
3. 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 15 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.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting. Do not attempt to break up cell clumps. 
5. Add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 37°C.

Subcultivation Ratio: 1:2 to 1:6.

Marcovici M, Prier JE. Enhancement of St. Louis arbovirus plaque formation by neutral red. J. Virol. 2: 178-181, 1968. PubMed: 4911850

Melnick JL. Tissue culture techniques and their application to original isolation, growth, and assay of poliomyelitis and orphan viruses. Ann. N.Y. Acad. Sci. 61: 754-772, 1955. PubMed: 13340582

Wolf K, et al. Duck viral enteritis: microtiter plate isolation and neutralization test using the duck embryo fibroblast cell line. Avian Dis. 18: 427-434, 1974. PubMed: 4368600

Buynak EB, et al. Preparation and testing of duck embryo cell culture rubella vaccine. Am. J. Dis. Child. 118: 347-354, 1969. PubMed: 4307520

Farris AD, et al. Conserved features of Y RNAs revealed by automated phylogenetic secondary structure analysis. Nucleic Acids Res. 27: 1070-1078, 1999. PubMed: 9927741

Nucleotide (GenBank) : U82124 Anas platyrhynchus Y1 RNA, partial sequence.

Nucleotide (GenBank) : U82125 Anas platyrhynchus Y3 RNA, partial sequence.