macrophage macrophage (alveolar); immortalized with SV40 large T antigen transformed with pSV3-neo
Product Format
frozen
Morphology
macrophage
Culture Properties
adherent
Biosafety Level
2 [Cells contain SV40 viral DNA sequences]
Age
27 days
Gender
unknown
Strain
Landrace
Applications
These porcine myelomonocytic cell lines may have a wide variety of applications in porcine virology and immunology Ref.
3D4/21 豬肺泡巨噬細胞
Storage Conditions
liquid nitrogen vapor phase
Derivation
The parental porcine monomyeloid cell line, 3D4, was established in December of 1998 following transfection of primary porcine alveolar macrophage cultures with the pSV3neo plasmid.
Single cell cloning and selection in G-418 of the 3D4 parental cell line resulted in establishment of 3D4/2 (
ATCC CRL-2845), 3D4/21 (ATCC CRL-2843) and 3D4/31 (ATCC CRL-2844).
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.
Complete Growth Medium
RPMI 1640 medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate supplemented with 0.1 mM nonessential amino acids, 90%; fetal bovine serum, 10%
Subculturing
Volumes used in this protocol are for 75cm2 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.
Subc*tion Ratio: A subc*tion 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.
Cryopreservation
Complete growth medium supplemented with 5% (v/v) DMSO. Cell culture tested DMSO is available as ATCC® Catalog No. 4-X.
