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3D4/2 (ATCC® CRL-2845™)

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 Ref.

Storage Conditions liquid nitrogen vapor phaseDerivation 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.

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℃ 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 3 x 103 to 6 x 103 viable cells/cm2 is recommended.

6，Incubate cultures at 37℃. Subculture when cell concentration reaches between 2 x 105 and 3 x 105cells/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 tested DMSO is available as ATCC® Catalog No. 4-X.

Culture Conditions Temperature: 37℃ Atmosphere: 5% CO2 in air