Comments |
The plasmid carries the genes for neomycin resistance and SV40 large T antigen.
A subpopulation of each cell line (3D4/2 (ATCC CRL-2845), 3D4/21 (ATCC CRL-2843) and 3D4/31 (ATCC CRL-2844)) was positive, to varying degrees depending on the media formulation, for nonspecific esterase activity and phagocytosis.
Clone 3D4/21 can produce Bovine adenovirus type 3 (BAV-3) to markedly higher titers than clones 3D4/2 and 3D4/31.
Addition of DMSO improved the capability of clone 3D4/21 to replicate the field isolate of African swine fever virus (ASFV/Lillie) compared to the other clones. |
Subculturing |
Volumes used in this protocol are for 75cm 2 flasks; 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 that 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 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.
- 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. An inoculum of 5 x 103 to 7 x 103 viable cells/cm2 is recommended.
- Incubate cultures at 37°C. Subculture when cell concentration reaches between 3 x 105 and 4 x 105 cells/cm2.
Subcultivation Ratio: A subcultivation ratio of 1:6 to 1:8 is recommended
Medium Renewal: Two to three times weekly
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture Of Animal Cells: A Manual Of Basic Technique by R. Ian Freshney, 5th edition, published by Wiley-Liss, N.Y., 2005. |
References |
Weingartl HM, et al. Continuous porcine cell lines developed from alveolar macrophages: partial characterization and virus susceptibility. J. Virol. Methods 104: 203-216, 2002. PubMed: 12088830
Hay, R. J., Caputo, J. L., and Macy, M. L., Eds. (1992), ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.
Caputo, J. L., Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988.
Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.
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