3D4/21 豬肺泡巨噬細胞
3D4/21 (ATCC^® CRL-2843

Organism	Sus scrofa, pig
Tissue	lung
Cell Type	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).
Virus Susceptibility	Bovine adenovirus 3 Classical swine fever virus , Classical swine fever virus Human parainfluenza virus 3 Swinepox virus Vesicular stomatitis New Jersey virus Porcine adenovirus Herpes simplex virus 1 African swine fever virus Pseudorabies virus Vaccinia virus Swine vesicular disease virus
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.

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).

Virus Susceptibility

Bovine adenovirus 3
Classical swine fever virus , Classical swine fever virus
Human parainfluenza virus 3
Swinepox virus
Vesicular stomatitis New Jersey virus
Porcine adenovirus
Herpes simplex virus 1
African swine fever virus
Pseudorabies virus
Vaccinia virus
Swine vesicular disease virus

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.

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 10⁵cells/cm². *Subction Ratio:** A subction 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.
Culture Conditions	Temperature: 37°C Atmosphere: 5% CO₂ in air recommended

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 10⁵cells/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.

Culture Conditions

Temperature: 37°C

Atmosphere: 5% CO₂ in air recommended

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3D4/21 (ATCC^® CRL-2843