| 產品名稱 |
CCD 1106 KERTr |
| 商品貨號 |
B164145 |
| Organism |
Homo sapiens, human |
| Tissue |
skin |
| Cell Type |
keratinocyte; human papillomavirus 16 (HPV-16) E6/E7 transformed |
| Product Format |
frozen |
| Morphology |
epithelial |
| Biosafety Level |
2
[Cells may contain the Human Papilloma viral (HPV) 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 |
133 days gestation |
| Storage Conditions |
liquid nitrogen vapor phase |
| Derivation |
Passage 2 cells were transformed with a retrovirus vector (LXSN16E6E7 produced by ATCC CRL-2203) in the presence of polybrene. |
| Antigen Expression |
epithelial specific antigen |
| Oncogene |
E6/E7 + |
| Genes Expressed |
E6/E7 +, epithelial specific antigen |
| Comments |
This line stains positively with antibody for cytokeratin.
After 50 population doublings, the cells continue dividing and retain cuboidal morphology.
E6E7 sequences were detected by PCR in cells at passage 18.
Major Histocompatibility Complex class I or II molecules were not expressed on these cells, but PCR analyses revealed presence of the genes for directing synthesis of HLA antigens.
Epithelial specific antigen was detected using antibody produced by the Ep16 hybridoma (ATCC HB-155). |
| Complete Growth Medium |
These cells are grown in Keratinocyte-Serum Free Medium (Gibco 17005-042) with added Keratinocytes Supplements (Gibco 37000-015) including Bovine Pituitary Extract (BPE; Gibco 13028-014) and human recombinant epidermal growth factor (EGF; Gibco 10450-013) further supplemented with:
- Additional 35 ng/mL human recombinant epidermal growth factor (EGF; BD cat# 354052).
Do not filter the complete medium.
This medium is formulated for use with a 5% CO2 in air atmosphere. |
| 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.
- 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.
- To remove trypsin-EDTA solution, transfer cell suspension to centrifuge tube 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.
- Place culture vessels in incubators at 37°C.
Subculture Ratio: 1:3 to 1:5
Medium Renewal: Twice a week.
Note: Add fresh medium twice per week
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994. |
| Cryopreservation |
Freeze Medium: Ham's F12 medium 85%; DMSO, 5%; FBS 10%
Storage Temperature: Liquid nitrogen vapor phase |
| Culture Conditions |
Temperature: 37°C |
| Name of Depositor |
L Vilner |
| Passage History |
E6E7 sequences were detected by PCR in cells at passage 18. Passage 2 cells were transformed with a retrovirus vector ( LXSN16E6E7 produced by ATCC CRL-2203) in the presence of polybrene. |
| References |
Zabrenetzky V, et al. The isolation, immortalization and characterization of human fetal keratinocytes. In Vitro Cell. Dev. Biol. 33: Part II, p. 34A, 1997.
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