產(chǎn)品名稱 |
PEAKrapid |
商品貨號 |
B214338 |
Organism |
Homo sapiens, human |
Tissue |
kidney |
Cell Type |
transformed with adenovirus 5 DNA |
Product Format |
frozen |
Morphology |
epithelial |
Culture Properties |
loosely adherent |
Biosafety Level |
2 [cells containing Adenovirus type 5 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 |
fetus |
Applications |
The cells are useful for protein expression. They support the replication of recombinant plasmids with the Epstein-Barr virus (EBV) oriP or SV40 origin of replication. |
Storage Conditions |
liquid nitrogen vapor phase |
Images |
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Derivation |
This line was derived from the human embryonic kidney line, HEK 293, ATCC CRL-1573. |
Complete Growth Medium |
Dulbecco's modified Eagle's medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose and supplemented with 0.05 mg/ml Gentamicin sulfate and 10% fetal bovine serum.
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Subculturing |
Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
Note: The cells are loosely adherent and will detach at room temperature.
- Remove culture medium to a centrifuge tube.
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Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53 mM EDTA solution to remove all traces of serum which 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 with 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.
- To remove trypsin-EDTA solution, transfer cell suspension to the centrifuge tube with the medium and cells from step #1 and spin at approximately 125 x g for 5 to 10 minutes.
- Discard supernatant and resuspend cells in fresh growth medium. Add appropriate aliquots of cell suspension to new culture vessels.
An inoculum of 5 x 103 to 5 x 104 viable cells/mL is recommended.
- Place culture vessels in incubators at 37°C.
Interval: Maintain cultures at a cell concentration between 8 x 103 and 1 x 105 cells/cm2.
Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:6 is recommended
Medium Renewal: Every two to three days
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 an additional 20% fetal bovine serum and 10% DMSO. Cell culture tested DMSO is available as ATCC® Catalog No. 4-X. |
Culture Conditions |
Temperature: 37°C
Atmosphere: Air, 95%; Carbon dioxide (CO2), 5% |
Population Doubling Time |
26 hours |
Name of Depositor |
J Seed |
References |
Nelson G, et al. An amino-acid taste receptor. Nature 416: 199-202, 2002. PubMed: 11894099
Magistrelli G, et al. Rapid, simple and high yield production of recombinant proteins in mammalian cells using a versatile episomal system. Protein Expression and Purification: 72(2): 209-216, 2010. PubMed: 20399863
Dobi A, et al. Mammalian expression cloning of nucleic acid binding proteins by agarose thin-layer gelshift clone selection. Biotechniques: 33(4): 868-872, 2002. PubMed: 12398195
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