人妻少妇偷人精品无码丨色婷婷av久久久久久久丨欧美xxxx做受性欧美88丨欧洲女人牲交视频免费丨亚洲精品久久久av无码专区

Note: 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

1. Daily maintain a sufficient number of flasks that have been pre-plated with MEFs in complete medium for feeder cells.
2. 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

1. Aspirate the medium from the flask(s) containing ES cells.
2. Wash with PBS Ca+2/Mg+2-free (ATCC® SCRR-2201).
3. 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.
4. 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.
5. Spin the cells at 270 x g for 5 min. Aspirate the supernatant.
6. 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.
7. 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.
8. 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.

Hug BA, et al. Analysis of mice containing a targeted deletion of beta-globin locus control region 5' hypersensitive site 3. Mol. Cell. Biol. 16: 2906-2912, 1996. PubMed: 8649401

Matise MP, et al. Production of targeted Embryonic Stem Cell Clones. In AL Joyner (Ed.), Gene Targeting: A Practical Approach. Oxford University Press, Oxford, p. 101-132, 1999.

Fabricius D, Bonde S, Zavazava N. Induction of stable mixed chimerism by embryonic stem cells requires functional Fas/FasL engagement. Transplantation 79(9): 1040-1044, 2005. PubMed: 15880040

Conte D, et al. Inhibitor of apoptosis protein cIAP2 is essential for lipopolysaccharide-induced macrophage survival. Mol. Cell Biol. 26(2): 699-708, 2006. PubMed: 16382159

Westervelt P, et al. High-penetrance mouse model of acute promyelocytic leukemia with very low levels of PML-RARalpha expression. Blood 102(5): 1857-1865, 2003. PubMed: 12750176

Schumacher A, et al. Staurosporine is a potent activator of neuronal, glial, and "CNS stem cell-like" neurosphere differentiation in murine embryonic stem cells. Mol. Cell Neurosci. 23: 669-680, 2003. PubMed: 12932446

Lee MH, et al. Simvastatin suppresses self-renewal of mouse embryonic stem cells by inhibiting RhoA geranylgeranylation. Stem Cells 25: 1654-1663, 2007. PubMed: 17464088