3D4/21 cell line猪肺巨噬细胞株细胞系 BioVector NTCC质粒载体菌种细胞基因保藏中心

3D4/21 cell line猪肺巨噬细胞株细胞系 BioVector NTCC质粒载体菌种细胞基因保藏中心
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] Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country. 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 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 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, 3D4/21 and 3D4/31) 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. 1. Remove and discard culture medium. 2. 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. 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. 5. 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. 6. 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. Cryopreservation Complete growth medium supplemented with 5% (v/v) DMSO. Cell Culture Conditions Temperature: 37°C Atmosphere: 5% CO2 in air recommended Population Doubling Time about 18 hrs
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