| 產品名稱 |
CE-1 |
| 商品貨號 |
B175588 |
| Organism |
Mus musculus, mouse |
| Tissue |
inner cell mass |
| Cell Type |
embryonic stem cell |
| Product Format |
frozen |
| Morphology |
spherical colony |
| Culture Properties |
adherent |
| Biosafety Level |
1
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 |
embryo |
| Strain |
129S2/SvPas |
| Storage Conditions |
liquid nitrogen vapor phase |
| Derivation |
The CE1 cell line was derived from the D3 ES cell line. [PubMed: 12591158] |
| Comments |
CE1 (for Cassette Exchange) contains one 'acceptor' module that allows for efficient double lox targeting. The cell line is also hygromycin resistant. The CE1 cell line was derived from the D3 ES cell line. [PubMed: 12591158] |
| Complete Growth Medium |
Grow ES cells in Mouse ES Cell Basal Medium (ATCC SCRR-2011) that has been supplemented with the following components:
1. 0.1 mM 2-mercaptoethanol (Life Technologies Cat. No. 21985-023)
2. 1,000 U/mL mouse leukemia inhibitory factor (LIF) (Millipore Cat. No. ESG1107)
3. 10% to 15% ES-Cell Qualified FBS (ATCC® SCRR-30-2020) or an ES cell qualified serum replacement
Complete Growth Medium for Mouse ES Cells is stable for 14 days when stored at 2°C to 8°C.
|
| Subculturing |
To insure the highest level of viability, pre-warm media and Trypsin/EDTA to 37ºC before adding to cells. Volumes used in this protocol are for T75 flasks. Proportionally adjust the volumes for culture vessels of other sizes. A split ratio of 1:4 to 1:7 is recommended.
Feeder Cell Preparation for Subcultures
- Daily maintain a sufficient number of flasks that have been pre-plated with MEFs in complete medium for feeder cells.
- One hour before subculturing the ES cells, perform a 100% medium change for the MEFs using complete growth medium for ES cells.
Dissociation and Transfer of ES Cells
- Aspirate the medium from the flask(s) containing ES cells.
- Wash with PBS Ca+2/Mg+2-free (ATCC® SCRR-2201).
- Add 3.0 mL of 0.25% (w/v) Trypsin / 0.53 mM EDTA solution (ATCC® 30-2101) and place in incubator. After about one minute the ES colonies will dissociate and all cells will detach from the flask.
- Dislodge the cells by gently tapping the side of the flask then wash the cells off with 7-10 mL of fresh culture medium. Triturate cells several times with a 10 mL pipette in order to dissociate the cells into a single-cell suspension.
- Spin the cells at 270 x g for 5 min. Aspirate the supernatant.
- Resuspend in enough complete growth medium for ES cells to reseed new vessels at the desired split ratio (i.e. a split ratio of 1:4 to 1:7 is recommended). Perform a cell count to determine the total number of cells. ES cells should be plated at a density of 30,000 – 50,000 cells/ cm2.
- Add separate aliquots of the cell suspension to the appropriate size flask containing feeder cells and add an appropriate volume of fresh complete growth medium for ES cells to each vessel.
- Incubate the culture at 37°C in a humidified 5% CO2/95% air incubator. Perform a 100% medium change every day, passage cells every 1-2 days.
|
| Cryopreservation |
Freeze medium: Complete growth medium supplemented with an additional 10% FBS and 10% DMSO.
Storage temperature: liquid nitrogen vapor phase |
| Culture Conditions |
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
Temperature: 37°C |
| Name of Depositor |
DI Gottlieb |
| Year of Origin |
September, 2001 |
| References |
Matise M, et alProduction of targeted embryonic stem cell clonesIn: Matise M, et alGene Targeting: A Practical ApproachOxfordOxford University Press101-132, 1999
Adams LD, et al. Double lox targeting for neural cell transgenesis. Brain Res. Mol. Brain Res. 110: 220-233, 2003. PubMed: 12591158
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